EP2970456A1 - Verfahren und zusammensetzungen zur abgabe von mrna-codierten antikörpern - Google Patents

Verfahren und zusammensetzungen zur abgabe von mrna-codierten antikörpern

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Publication number
EP2970456A1
EP2970456A1 EP14717945.1A EP14717945A EP2970456A1 EP 2970456 A1 EP2970456 A1 EP 2970456A1 EP 14717945 A EP14717945 A EP 14717945A EP 2970456 A1 EP2970456 A1 EP 2970456A1
Authority
EP
European Patent Office
Prior art keywords
mrna encoding
antibody
mrna
light chain
heavy chain
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
EP14717945.1A
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English (en)
French (fr)
Other versions
EP2970456B1 (de
Inventor
Michael Heartlein
Frank Derosa
Braydon Charles Guild
Anusha DIAS
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Translate Bio Inc
Original Assignee
Shire Human Genetics Therapies Inc
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Application filed by Shire Human Genetics Therapies Inc filed Critical Shire Human Genetics Therapies Inc
Priority to SI201431860T priority Critical patent/SI2970456T1/sl
Priority to HRP20211119TT priority patent/HRP20211119T1/hr
Priority to EP21170989.4A priority patent/EP3932947A1/de
Priority to RS20210906A priority patent/RS62565B1/sr
Priority to PL14717945T priority patent/PL2970456T3/pl
Publication of EP2970456A1 publication Critical patent/EP2970456A1/de
Application granted granted Critical
Publication of EP2970456B1 publication Critical patent/EP2970456B1/de
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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/22—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against growth factors ; against growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/88—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation using microencapsulation, e.g. using amphiphile liposome vesicle
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature

Definitions

  • Antibodies are known to have powerful therapeutic effects and are currently used for the treatment of a range of diseases including cancer, autoimmune diseases, cardiovascular disease, and transplant rejection.
  • therapeutic antibodies are produced by recombinant technology, formulated and then administered to patients in need of antibody therapy.
  • antibody production and formulation is highly expensive.
  • many antibodies only have a very short half-life in vivo and therefore, may not reach their target antigen or target tissue before being degraded.
  • antibody therapy often requires high doses and frequent administration.
  • Gene therapy and genetic vaccination provide alternative approaches for delivery of large amounts of antibodies in vivo.
  • DNA is degraded slowly in the bloodstream. Formation of anti-DNA antibodies may occur (Gilkeson et al, J. Clin. Invest. 1995, 95: 1398-1402). The possible persistence of (foreign) DNA in the organism can thus lead to a hyperactivation of the immune system, which was known to result in splenomegaly in mice (Montheith et al., Anticancer Drug Res. 1997, 12(5): 421-432).
  • DNA integration can cause mutations in the host genome by interrupting an intact gene.
  • the present invention provides an improved method for safer and more effective delivery of antibodies in vivo based on messenger RNA (mRNA) delivery technology.
  • mRNA messenger RNA
  • the present invention is, in part, based on the surprising discovery that production of fully assembled multi-chain antibodies can be accomplished in vivo by delivering exogenous mRNAs encoding a heavy chain and a light chain of the antibody, even when the heavy chain and light chain are delivered by separate mRNAs.
  • mRNA messenger RNA
  • the present invention provides a method of delivering an antibody in vivo, by administering to a subject in need thereof one or more mRNAs encoding a heavy chain and a light chain of an antibody, and wherein the antibody is expressed systemically in the subject.
  • the one or more mRNAs comprise a first mRNA encoding the heavy chain and a second mRNA encoding the light chain of the antibody.
  • the one or more mRNAs comprise a single mRNA encoding both the heavy chain and the light chain of the antibody.
  • a heavy chain or light chain encoding mRNA comprises a sequence encoding a signal peptide. In some embodiments, a heavy chain or light chain encoding mRNA comprises a sequence encoding a human growth hormone (hGH) signal peptide
  • hGH human growth hormone
  • sequence encoding a signal peptide sequence (e.g., SEQ ID N0:9 or SEQ ID NO: 10) is linked, directly or indirectly, to the heavy chain or light chain encoding mRNA sequence at the N-terminus.
  • the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio ranging between approximately 10: 1 to 1 : 10 (e.g., between approximately 9:1 to 1 :9, 8: 1 to 1 :8, 7: 1 to 1 :7, 6: 1 to 1 :6, 5:1 to 1 :5, 4: 1 to 1 :4, 3 : 1 to 1 :3, or 2: 1 to 1 :2).
  • the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio ranging between
  • the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio of approximately 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3:1, 2: 1 or 1 : 1. In some embodiments, the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio of approximately 4: 1. In some embodiments, the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio of approximately 1 : 1.
  • the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are present at a ratio greater than 1 (e.g. , ranging between approximately 10: 1 to 1 : 1, 9: 1 to 1 : 1, 8: 1 to 1 : 1, 7: 1 to 1 : 1, 6: 1 to 1 : 1, 5: 1 to 1 : 1, 4: 1 to 1 : 1, 3: 1 to 1 : 1, or 2: 1 to 1 : 1).
  • 1 e.g. , ranging between approximately 10: 1 to 1 : 1, 9: 1 to 1 : 1, 8: 1 to 1 : 1, 7: 1 to 1 : 1, 6: 1 to 1 : 1, 5: 1 to 1 : 1, 4: 1 to 1 : 1, 3: 1 to 1 : 1, or 2: 1 to 1 : 1).
  • the one or more mRNAs encoding the heavy chain and the light chain of the antibody are delivered via a polymer and/or lipid based delivery vehicle. In some embodiments, the one or more mRNAs encoding the heavy chain and the light chain of the antibody are encapsulated within one or more liposomes. In some embodiments, the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are encapsulated in separate liposomes. In some embodiments, the first mRNA encoding the heavy chain and the second mRNA encoding the light chain are encapsulated in the same liposome.
  • the one or more liposomes comprise one or more of cationic lipid, neutral lipid, cholesterol-based lipid, and PEG-modified lipid. In some embodiments, the one or more liposomes comprise cationic lipid, neutral lipid, cholesterol-based lipid, and PEG-modified lipid.
  • the one or more liposomes have a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, the one or more liposomes have a size no greater than about 150 nm. In some embodiments, the one or more liposomes have a size no greater than about 100 nm. In some embodiments, the one or more liposomes have a size no greater than about 75 nm. In some embodiments, the one or more liposomes have a size no greater than about 50 nm.
  • the one or more liposomes have a size ranging from about
  • the one or more liposomes have a size ranging from about 250 - 100 nm (e.g., ranging from about 225 - 100 nm, 200 - 100 nm, 175 - 100 nm, 150 - 100 nm).
  • the one or more liposomes have a size ranging from about 250 - 100 nm (e.g., ranging from about 225 - 100 nm, 200 - 100 nm, 175 - 100 nm, 150 - 100 nm).
  • the one or more liposomes have a size ranging from about 100 - 10 nm (e.g., ranging from about 90 - 10 nm, 80 - 10 nm, 70 - 10 nm, 60 - 10 nm, or 50 - 10 nm).
  • the one or more mRNAs are modified to enhance stability.
  • the one or more mRNAs are modified to include a modified nucleotide, a modified sugar backbone, a cap structure, a poly A tail, a 5' and/or 3' untranslated region. In some embodiments, the one or more mRNAs are unmodified.
  • the one or more mRNAs are administered intravenously.
  • the one or more mRNAs are administered intraperitoneally. In some embodiments, the one or more mRNAs are administered subcutaneously. In some embodiments, the one or more mRNAs are administered via pulmonary administration.
  • the systemic expression of the antibody is detectable at least about 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 96 hours, 120 hours, 144 hours, 156 hours, 168 hours, or 180 hours post-administration (e.g., post single administration). In some embodiments, the systemic expression of the antibody is detectable at least about 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 20 days, 22 days, 25 days, or 30 days post-administration (e.g., post single administration).
  • the systemic expression of the antibody is detectable at least about 0.5 weeks, 1 week, 1.5 weeks, 2 weeks, 2.5 weeks, 3 weeks, 3.5 weeks, 4 weeks, 4.5 weeks, 5 weeks, 5.5 weeks, 6 weeks, 6.5 weeks, 7 weeks, 7.5 weeks, or 8 weeks post-administration (e.g., post single administration). In some embodiments, the systemic expression of the antibody is detectable at least about 1 month, 2 months, 3 months, or 4 months post-administration (e.g., post single administration).
  • the antibody is an intact immunoglobulin, (Fab) 2 , (Fab') 2 ,
  • the antibody is an IgG.
  • the antibody is selected from the group consisting of anti-CCL2, anti-lysyl oxidase-like-2 (LOXL2), anti-Fit- 1, anti-TNF-a, anti-Interleukin-2Ra receptor (CD25), anti-TGF , anti-B-cell activating factor, anti-alpha-4 integrin, anti-BAGE, anti-P-catenin/m, anti-Bcr-abl, anti-C5, anti-CA125, anti-CAMEL, anti-CAP-1, anti-CASP-8, anti-CD4, anti-CD19, anti-CD20, anti-CD22, anti- CD25, anti-CDC27/m, anti-CD 30, anti-CD33, anti-CD52, anti-CD56, anti-CD80, anti-CDK4/m, anti-CEA, anti-CT, anti-CTL4, anti-Cyp-B, anti-DAM,
  • LXL2 anti-lysyl oxidase-like-2
  • the present invention provides a method of producing an antibody by administering to a cell a first mRNA encoding a heavy chain and a second mRNA encoding a light chain of an antibody, and wherein the antibody is produced by the cell.
  • the cell is a mammalian cell.
  • the cell is a human cell.
  • the cell is a cultured cell.
  • the cell is a cell within a living organism.
  • the antibody is expressed intracellularly.
  • the antibody is secreted by the cell.
  • compositions including a first mRNA encoding a heavy chain and a second mRNA encoding a light chain of an antibody, wherein the first mRNA and the second mRNA are encapsulated in one or more liposomes.
  • the present invention also provides exemplary mRNAs encoding a heavy chain and a light chain of specific antibodies such as, for example, an anti- CCL2 antibody, and compositions containing the same.
  • the present invention provides an mRNA encoding a heavy chain of an anti-CCL2 antibody having a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:l or SEQ ID NO:2, as described herein.
  • the present invention provides an mRNA encoding a heavy chain of an anti-CCL2 antibody having a sequence of SEQ ID NO: l or SEQ ID NO:2, as described herein.
  • the present invention provides an mRNA encoding a light chain of an anti-CCL2 antibody having a sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:3 or SEQ ID NO:4, as described herein.
  • the present invention provides an mRNA encoding a light chain of an anti-CCL2 antibody having a sequence of SEQ ID NO:3 or SEQ ID NO:4, as described herein.
  • Figure 1 shows an exemplary bar graph of IgG protein levels, as determined by
  • Figure 2 shows an exemplary bar graph of IgG protein levels, as determined by
  • Figure 3 depicts the results of a western blot examining protein levels resulting from introduction of mRNA, according to provided methods, in HCL1 cells after 24 and 48 hours.
  • Figure 4 shows an exemplary bar graph of CCL2 antibody levels as determined via ELISA in the serum of mice exposed to mRNA according to provided methods for 6, 24, 48, or 72 hours.
  • Figure 5 shows an exemplary bar graph of a-VEGF antibody levels as determined via ELISA in the serum of mice after single dose of a-VEGF mRNA.
  • Figure 6 shows an exemplary bar graph of a-VEGF antibody levels as determined via ELISA in the serum of individually identified mice after single dose of a-VEGF mRNA.
  • Figure 7 shows an exemplary bar graph of in vivo production of an anti-human
  • VEGF antibody in wild type mice 24 hours after dosing with a-VEGF mRNA loaded cK -E12 lipid nanoparticles (LNP). Mice were dosed via either tail vein injection or subcutaneous (SC) injection.
  • LNP cK -E12 lipid nanoparticles
  • amino acid in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain.
  • an amino acid has the general structure H 2 N-C(H)(R)-COOH.
  • an amino acid is a naturally occurring amino acid.
  • an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid.
  • Standard amino acid refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • synthetic amino acid encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions.
  • Amino acids, including carboxy- and/or amino-terminal amino acids in peptides can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond.
  • Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.).
  • chemical entities e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.
  • amino acid is used interchangeably with "amino acid residue,” and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a
  • animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans, at any stage of development. In some embodiments, “animal” refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In some embodiments, an animal may be a transgenic animal, genetically-engineered animal, and/or a clone.
  • mammal e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig.
  • Antibody encompasses both intact antibody and antibody fragment. Typically, an intact “antibody” is an immunoglobulin that binds specifically to a particular antigen.
  • An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgE, and IgD.
  • a typical immunoglobulin (antibody) structural unit as understood in the art, is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (approximately 25 kD) and one "heavy” chain (approximately 50-70 kD).
  • each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • the terms "variable light chain”(VL) and “variable heavy chain” (VH) refer to these light and heavy chains respectively.
  • Each variable region is further subdivided into hypervariable (HV) and framework (FR) regions.
  • the hypervariable regions comprise three areas of hypervariability sequence called complementarity determining regions (CDR 1, CDR 2 and CDR 3), separated by four framework regions (FR1, FR2, FR2, and FR4) which form a beta-sheet structure and serve as a scaffold to hold the HV regions in position.
  • each heavy and light chain defines a constant region consisting of one domain for the light chain (CL) and three for the heavy chain (CHI, CH2 and CH3).
  • the terms "intact antibody” or “fully assembled antibody” are used in reference to an antibody to mean that it contains two heavy chains and two light chains, optionally associated by disulfide bonds as occurs with naturally-produced antibodies.
  • an antibody according to the present invention is an antibody fragment.
  • an "antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments.
  • antibody fragments include isolated fragments, "Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • an antibody fragment contains sufficient sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some
  • a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen.
  • antigen binding fragments of an antibody include, but are not limited to, Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment, and an isolated complementarity determining region (CDR) region.
  • Bioavailability As used herein, the term “bioavailability” generally refers to the percentage of the administered dose that reaches the blood stream of a subject.
  • biologically active refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.
  • expression refers to translation of an m NA into a polypeptide (e.g., heavy chain or light chain of antibody), assemble multiple polypeptides (e.g., heavy chain or light chain of antibody) into an intact protein (e.g., antibody) and/or post-translational modification of a polypeptide or fully assembled protein (e.g., antibody).
  • polypeptide e.g., heavy chain or light chain of antibody
  • assemble multiple polypeptides e.g., heavy chain or light chain of antibody
  • an intact protein e.g., antibody
  • post-translational modification of a polypeptide or fully assembled protein e.g., antibody
  • a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
  • GC content is the fraction or percentage of total nucleobase residues in a nucleic acid sequence that are guanine residues, cytosine residues, or analogs thereof. For example, a 100 nt sequence that contains exactly 30 cytosines, exactly 30 guanines, exactly one cytosine analog, and exactly one guanine analog has a GC richness of 62%.
  • Half-life As used herein, the term "half-life" is the time required for a quantity such as protein concentration or activity to fall to half of its value as measured at the beginning of a time period.
  • “reduce,” or grammatical equivalents indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein.
  • a “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.
  • in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.
  • m vivo refers to events that occur within a multi-cellular organism, such as a human and a non-human animal. In the context of cell-based systems, the term may be used to refer to events that occur within a living cell (as opposed to, for example, in vitro systems).
  • Isolated refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%o, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated.
  • isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%o, about 98%, about 99%, or more than about 99% pure.
  • a substance is "pure” if it is substantially free of other components.
  • calculation of percent purity of isolated substances and/or entities should not include excipients ⁇ e.g., buffer, solvent, water, etc.).
  • Linker refers to, in a fusion protein, an amino acid sequence other than that appearing at a particular position in the natural protein and is generally designed to be flexible or to interpose a structure, such as an a-helix, between two protein moieties.
  • a linker is also referred to as a spacer.
  • a linker or a spacer typically does not have biological function on its own.
  • tissue specific delivery or distribution refers to tissue specific delivery or distribution.
  • mR protein encoded by mR As
  • messenger RNA As used herein, the term "messenger RNA (mR A)" refers to a polynucleotide that encodes at least one polypeptide. mRNA as used herein encompasses both modified and unmodified RNA. mRNA may contain one or more coding and non-coding regions.
  • nucleic acid refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain.
  • a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage.
  • nucleic acid refers to individual nucleic acid residues ⁇ e.g., nucleotides and/or nucleosides).
  • nucleic acid refers to a polynucleotide chain comprising individual nucleic acid residues.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • nucleic acid encompasses RNA as well as single and/or double-stranded DNA and/or cDNA.
  • nucleic acid “DNA,” “RNA,” and/or similar terms include nucleic acid analogs, i.e., analogs having other than a
  • nucleic acids which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc.
  • nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc.
  • a nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated.
  • a nucleic acid is or comprises natural nucleosides ⁇ e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2- thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaa
  • the present invention is specifically directed to "unmodified nucleic acids,” meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.
  • nucleic acids e.g., polynucleotides and residues, including nucleotides and/or nucleosides
  • patient refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes.
  • Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans).
  • animals e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans.
  • a patient is a human.
  • a human includes pre and post natal forms.
  • compositions that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
  • systemic delivery refers to a delivery or distribution mechanism or approach that affect the entire body or an entire organism.
  • systemic distribution or delivery is accomplished via body's circulation system, e.g., blood stream.
  • Subject refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate).
  • a human includes pre- and post-natal forms.
  • a subject is a human being.
  • a subject can be a patient, which refers to a human presenting to a medical provider for diagnosis or treatment of a disease.
  • the term "subject” is used herein interchangeably with “individual” or "patient.”
  • a subject can be afflicted with or is susceptible to a disease or disorder but may or may not display symptoms of the disease or disorder.
  • the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
  • One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
  • the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
  • Target tissues refers to any tissue that is affected by a disease to be treated.
  • target tissues include those tissues that display disease-associated pathology, symptom, or feature.
  • therapeutically effective amount As used herein, the term "therapeutically effective amount" of a therapeutic agent means an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, diagnose, prevent, and/or delay the onset of the symptom(s) of the disease, disorder, and/or condition. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.
  • Treating refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease. DETAILED DESCRIPTION
  • the present invention provides, among other things, methods and compositions for delivering antibodies in vivo based on mRNA delivery technology.
  • the present invention provides a method of delivery an antibody by administering to a subject in need of delivery one or more mRNAs encoding a heavy chain and a light chain of the antibody.
  • the heavy chain and the light chain of an antibody are delivered by separate mRNAs.
  • the heavy chain and the light chain of an antibody are delivered by a same mRNA.
  • mRNAs may be delivered as packaged particles (e.g., encapsulated in liposomes or polymer based vehicles) or unpackaged (i.e., naked).
  • mRNA encoded antibodies may be expressed locally (e.g., in a tissue specific manner) or systematically in the subject.
  • the present invention may be used to deliver any type of antibodies.
  • the term "antibody” encompasses both intact antibody and antibody fragment. Typically, an intact “antibody” is an immunoglobulin that binds specifically to a particular antigen. An antibody may be a member of any immunoglobulin class, including any of the human
  • an intact antibody is a tetramer.
  • Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light"
  • variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • VL variable light chain
  • VH variable heavy chain
  • Each variable region can be further subdivided into hypervariable (HV) and framework (FR) regions.
  • the hypervariable regions comprise three areas of hypervariability sequence called
  • CDR 1, CDR 2 and CDR 3 complementarity determining regions
  • FR1, FR2, FR2, and FR4 framework regions which form a beta-sheet structure and serve as a scaffold to hold the HV regions in position.
  • the C-terminus of each heavy and light chain defines a constant region consisting of one domain for the light chain (CL) and three for the heavy chain (CHI, CH2 and CH3).
  • a light chain of immunoglobulins can be further differentiated into the isotypes kappa and lamda.
  • an antibody according to the present invention is an antibody fragment.
  • an "antibody fragment” includes a portion of an intact antibody, such as, for example, the antigen-binding or variable region of an antibody.
  • antibody fragments include Fab, Fab', F(ab') 2 , and Fv fragments; triabodies; tetrabodies; linear antibodies; single-chain antibody molecules; and multi specific antibodies formed from antibody fragments.
  • antibody fragments include isolated fragments, "Fv” fragments, consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy chain variable regions are connected by a peptide linker ("ScFv proteins"), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • an antibody fragment contains a sufficient sequence of the parent antibody of which it is a fragment that it binds to the same antigen as does the parent antibody; in some embodiments, a fragment binds to the antigen with a comparable affinity to that of the parent antibody and/or competes with the parent antibody for binding to the antigen.
  • antigen binding fragments of an antibody include, but are not limited to, Fab fragment, Fab' fragment, F(ab') 2 fragment, scFv fragment, Fv fragment, dsFv diabody, dAb fragment, Fd' fragment, Fd fragment, and an isolated complementarity determining region (CDR).
  • the present invention may be used to deliver any antibody known in the art and antibodies that can be produced against desired antigens using standard methods.
  • the present invention may be used to deliver monoclonal antibodies, polyclonal antibodies, antibody mixtures or cocktails, human or humanized antibodies, chimeric antibodies, or bi-specific antibodies.
  • Exemplary antibodies include, but are not limited to, anti-chemokine (C-C motif) ligand 2 (CCL2), , anti-lysyl oxidase-like-2 (LOXL2), anti-Fit- 1, anti-TNF-a, anti-Interleukin- 2Ra receptor (CD25), anti-TGF , anti-B-cell activating factor, anti-alpha-4 integrin, anti-BAGE, anti-P-catenin/m, anti-Bcr-abl, anti-C5, anti-CA125, anti-CAMEL, anti-CAP-1, anti-CASP-8, anti-CD4, anti-CD19, anti-CD20, anti-CD22, anti-CD25, anti-CDC27/m, anti-CD 30, anti-CD33, anti-CD52, anti-CD56, anti-CD80, anti-CDK4/m, anti-CEA, anti-CT, anti-CTL4, anti-Cyp-B, anti-DAM, anti-EGFR, anti-Er
  • antibodies may be produced in a cell or living organism through exogenous mRNA translation inside the cell and living organism.
  • production of fully assembled multi-chain antibodies can be accomplished in a cell or living organism by delivering exogenous mRNAs encoding a heavy chain and a light chain of the antibody.
  • a tetramer containing two heavy chains and two light chains is produced.
  • the term "heavy chain” encompasses all types of naturally- occurring heavy chains of different classes of immunoglobulins, including but not limited to, 3 ⁇ 4 ⁇ ( ⁇ ), IgD ( ⁇ ), IgG(y), IgA(a), and IgE(s), and biologically active variants thereof.
  • a heavy chain according to the present invention contains the N-terminal variable region responsible for antigen recognition, typically including CDR 1, CDR 2 and CDR 3, separated by four framework regions (FR1, FR2, FR2, and FR4).
  • the N-terminal variable region contains about 100 to 110 or more amino acids.
  • a heavy chain according to the present invention contains one or more of constant domains (e.g., C H I , C H 2, and/or C H 3).
  • an mRNA encoding a heavy chain of an antibody is of or greater than 0.3 kb, 0.5 kb, 0.75 kb, 1.0 kb, 1.25 kb, 1.5 kb, 1.75 kb, 2.0 kb, 2.5 kb, 3.0 kb, 3.5 kb, 4.0 kb in length.
  • a light chain encompasses all types of naturally- occurring light chains of different classes of immunoglobulins, including but not limited to ⁇ or ⁇ isotypes, and biologically active variants thereof.
  • a light chain according to the present invention comprises an N-terminal variable domain (V L ).
  • a light chain according to the present invention contains a C-terminal constant domain (C L ).
  • an mRNA encoding a light chain of an antibody is of or greater than 0.1 kb, 0.2 kb, 0.3 kb, 0.4 kb, 0.5 kb, 0.6 kb, 0.7 kb, 0.8 kb, 0.9 kb, 1.0 kb, 1.25 kb, 1.5 kb, 1.75 kb, 2.0 kb, 2.5 kb, or 3.0 kb in length.
  • a tetrameric antibody containing two heavy chains and two light chains encoded by mRNAs, each bonded together by a disulfide bridge.
  • a heavy chain and light chain of an antibody may be encoded and delivered by a single mRNA or separate mRNAs. It is contemplated that it may be advantageous to deliver heavy chain encoding mRNA and light chain encoding mRNA at varying ratios in order to optimize production of fully assembled functional antibodies.
  • the heavy chain encoding mRNA also referred to as the first mRNA
  • the light chain encoding mRNA also referred to as the second mRNA
  • the heavy chain encoding mRNA are delivered at a ratio ranging between approximately 10: 1 to 1 : 10 (e.g., between approximately 9:1 to 1 :9, 8: 1 to 1 :8,
  • the heavy chain encoding mRNA also referred to as the first mRNA
  • the light chain encoding mRNA also referred to as the second mRNA
  • the heavy chain encoding mRNA and the light chain encoding mRNA are delivered at a ratio of or greater than approximately 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1 or 1 : 1.
  • the heavy chain encoding mRNA also referred to as the first mRNA
  • the light chain encoding mRNA also referred to as the second mRNA
  • the heavy chain encoding mRNA and the light chain encoding mRNA are delivered at a ratio other than 1 : 1 (equal molar).
  • the heavy chain encoding mRNA also referred to as the first mRNA
  • the light chain encoding mRNA also referred to as the second mRNA
  • the ratio greater than 1 e.g., ranging between approximately 10:1 to 1:1, 9:1 to 1:1, 8:1 to 1:1, 7:1 to 1:1, 6:1 to 1:1, 5:1 to 1:1,4:1 to 1:1, 3:1 to 1:1, or 2:1 to 1:1.
  • the heavy chain encoding mRNA also referred to as the first mRNA
  • the light chain encoding mRNA also referred to as the second mRNA
  • the heavy chain encoding mRNA and the light chain encoding mRNA are delivered at a ratio less than 1 (e.g., ranging between approximately 1:1 to 1:10, 1:1 to 1:9, 1:1 to 1:8, 1:1 to 1:7, 1:1 to 1:6, 1:1 to 1:5, 1:1 to 1:4, 1:1 to 1:3, or 1:1 to 1:2).
  • an mRNA encoding a heavy chain and/or light chain incorporates a nucleotide sequence encoding a signal peptide.
  • signal peptide refers to a peptide present at a newly synthesized protein that can target the protein towards the secretory pathway. Typically, the signal peptide is cleaved after translocation into the endoplasmic reticulum following translation of the mRNA. Signal peptide is also referred to as signal sequence, leader sequence or leader peptide. Typically, a signal peptide is a short (e.g., 5-30, 5-25, 5-20, 5-15, or 5-10 amino acids long) peptide.
  • a signal peptide may be present at the N-terminus of a newly synthesized protein.
  • the incorporation of a signal peptide encoding sequence on a heavy chain and/or light chain encoding mRNA may facilitate the secretion and/or production of the antibody produced from the mRNA in vivo.
  • a suitable signal peptide for the present invention can be a heterogeneous sequence derived from various eukaryotic and prokaryotic proteins, in particular secreted proteins.
  • a suitable signal peptide is a leucine-rich sequence. See Yamamoto Y et al. (1989), Biochemistry, 28:2728-2732, which is incorporated herein by reference.
  • a suitable signal peptide may be derived from a human growth hormone (hGH), serum albumin preproprotein, Ig kappa light chain precursor, Azurocidin preproprotein, cystatin- S precursor, trypsinogen 2 precursor, potassium channel blocker, alpha conotoxin lpl.3, alpha conotoxin, alfa-galactosidase, cellulose, aspartic proteinase nepenthesin-1, acid chitinase, K28 prepro-toxin, killer toxin zygocin precursor, and Cholera toxin.
  • Exemplary signal peptide sequences are described in Kober, et al, Biotechnol.
  • a heavy chain and/or light chain encoding mR A may incorporate a sequence encoding a signal peptide derived from human growth hormone (hGH), or a fragment thereof.
  • hGH human growth hormone
  • a non-limiting nucleotide sequence encoding a hGH signal peptide is show below.
  • hGH human growth hormone
  • hGH human growth hormone
  • an mRNA according to the present invention may incorporate a signal peptide encoding sequence having at least 50%, 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to SEQ ID NO:9 or SEQ ID NO: 10.
  • mRNAs according to the present invention may be synthesized according to any of a variety of known methods.
  • mRNAs according to the present invention may be synthesized via in vitro transcription (IVT).
  • IVT in vitro transcription
  • IVT is typically performed with a linear or circular DNA template containing a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and/or RNAse inhibitor.
  • RNA polymerase e.g., T3, T7 or SP6 RNA polymerase
  • a DNA template is transcribed in vitro.
  • a suitable DNA template typically has a promoter, for example a T3, T7 or SP6 promoter, for in vitro transcription, followed by desired nucleotide sequence for desired antibody encoding (e.g., heavy chain or light chain encoding) mRNA and a termination signal.
  • Desired antibody encoding (e.g., heavy chain or light chain encoding) mR A sequence according to the invention may be determined and incorporated into a DNA template using standard methods.
  • a virtual reverse translation is carried out based on the degenerated genetic code. Optimization algorithms may then be used for selection of suitable codons.
  • the G/C content can be optimized to achieve the highest possible G/C content on one hand, taking into the best possible account the frequency of the tRNAs according to codon usage on the other hand.
  • the optimized RNA sequence can be established and displayed, for example, with the aid of an appropriate display device and compared with the original (wild-type) sequence.
  • a secondary structure can also be analyzed to calculate stabilizing and destabilizing properties or, respectively, regions of the RNA.
  • mRNA according to the present invention may be synthesized as unmodified or modified mRNA.
  • mRNAs are modified to enhance stability.
  • Modifications of mRNA can include, for example, modifications of the nucleotides of the RNA.
  • a modified mRNA according to the invention can thus include, for example, backbone modifications, sugar modifications or base modifications.
  • antibody encoding mRNAs may be synthesized from naturally occurring nucleotides and/or nucleotide analogues (modified nucleotides) including, but not limited to, purines (adenine (A), guanine (G)) or pyrimidines (thymine (T), cytosine (C), uracil (U)), and as modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • nucleotide analogues modified nucleotides
  • purines adenine (A), guanine (G)
  • pyrimidines thymine (T), cytosine (C), uracil (U)
  • modified nucleotides analogues or derivatives of purines and pyrimidines, such as e.g.
  • antibody encoding mRNAs may contain RNA backbone modifications.
  • a backbone modification is a modification in which the phosphates of the backbone of the nucleotides contained in the RNA are modified chemically.
  • Exemplary backbone modifications typically include, but are not limited to, modifications from the group consisting of methylphosphonates, methylphosphoramidates, phosphoramidates, phosphorothioates (e.g. cytidine 5'-0-(l- thiophosphate)), boranophosphates, positively charged guanidinium groups etc., which means by replacing the phosphodiester linkage by other anionic, cationic or neutral groups.
  • antibody encoding mRNAs may contain sugar modifications.
  • a typical sugar modification is a chemical modification of the sugar of the nucleotides it contains including, but not limited to, sugar modifications chosen from the group consisting of 2'-deoxy-2'-fluoro-oligoribonucleotide (2'-fluoro-2'-deoxycytidine 5 '-triphosphate, 2'-fluoro-2'-deoxyuridine 5'-triphosphate), 2'-deoxy- 2'-deamine-oligoribonucleotide (2'-amino-2'-deoxy cytidine 5 '-triphosphate, 2'-amino-2'- deoxyuridine 5'-triphosphate), 2'-0-alkyloligoribonucleotide, 2'-deoxy-2'-C- alkyloligoribonucleotide (2'-0-methylcytidine 5'-triphosphat
  • antibody encoding mRNAs may contain modifications of the bases of the nucleotides (base modifications).
  • base modifications A modified nucleotide which contains a base modification is also called a base- modified nucleotide.
  • base-modified nucleotides include, but are not limited to,
  • mRNA synthesis includes the addition of a "cap" on the N-terminal
  • antibody encoding mRNAs include a 5' cap structure.
  • a 5' cap is typically added as follows: first, an RNA terminal phosphatase removes one of the terminal phosphate groups from the 5 ' nucleotide, leaving two terminal phosphates; guanosine triphosphate (GTP) is then added to the terminal phosphates via a guanylyl transferase, producing a 5 '5 '5 triphosphate linkage; and the 7-nitrogen of guanine is then methylated by a methyltransferase.
  • GTP guanosine triphosphate
  • cap structures include, but are not limited to, m7G(5')ppp (5'(A,G(5')ppp(5')A and G(5')ppp(5')G.
  • antibody encoding mRNAs include a 3' poly(A) tail structure.
  • a poly- A tail on the 3' terminus of mRNA typically includes about 10 to 300 adenosine nucleotides (e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nucleotides, about about 10 to 125 adenosine nucleotides, 10 to 100 adenosine nucleotides, about 10 to 75 adenosine nucleotides, about 20 to 70 adenosine nucleotides, or about 20 to 60 adenosine nucleotides).
  • adenosine nucleotides e.g., about 10 to 200 adenosine nucleotides, about 10 to 175 adenosine nucleotides, about 10 to 150 adenosine nu
  • antibody encoding mRNAs include a 3' poly(C) tail structure.
  • a suitable poly-C tail on the 3' terminus of mRNA typically include about 10 to 200 cytosine nucleotides (e.g., about 10 to 150 cytosine nucleotides, about 10 to 100 cytosine nucleotides, about 20 to 70 cytosine nucleotides, about 20 to 60 cytosine nucleotides, or about 10 to 40 cytosine nucleotides).
  • the poly-C tail may be added to the poly-A tail or may substitute the poly-A tail.
  • antibody encoding mR As include a 5' and/or 3' untranslated region.
  • a 5' untranslated region includes one or more elements that affect an mRNA's stability or translation, for example, an iron responsive element.
  • a 5' untranslated region may be between about 50 and 500 nucleotides in length (e.g., about 50 and 400 nucleotides in length, about 50 and 300 nucleotides in length, about 50 and 200 nucleotides in length, or about 50 and 100 nucleotides in length).
  • a 5' region of antibody encoding mRNAs includes a sequence encoding a signal peptide, such as those described herein.
  • a signal peptide derived from human growth hormone (hGH) e.g. SEQ ID NO:9 is incorporated in the 5' region.
  • hGH human growth hormone
  • a signal peptide encoding sequence e.g., hGH signal peptide encoding sequence such as SEQ ID NO:9 is linked, directly or indirectly, to the heavy chain or light chain encoding sequence at the N- terminus.
  • mRNAs encoding the heavy chain and light chain of an anti-CCL2 antibody are described in Example 1.
  • the heavy chain encoding mRNA without and with the 5' and 3' UTR sequences are shown below as SEQ ID NO: l and SEQ ID NO:2, respectively.
  • the light chain encoding mRNA without and with the 5 ' and 3 ' UTR sequences are shown below as SEQ ID NO:3 and SEQ ID NO:4, respectively.
  • Heavy chain anti-CCL2 (HC-aCCL2) mRNA without 5 ' and 3 ' UTR (SEQ ID NO:l):
  • Heavy chain anti-CCL2 HC-aCCL2 niRNA with 5 ' and 3 ' UTR (SEQ ID NO: 2):
  • LC-aCCL2 Light chain anti-CCL2 mRNA without 5 ' and 3 ' UTR
  • LC-aCCL2 Light chain anti-CCL2 mRNA with 5 ' and 3 ' UTR (SEQ ID NO:4):
  • the present invention also provides m NAs encoding a heavy chain and light chain of an anti-CCL2 antibody substantially identical or similar to the sequences described herein.
  • an mR A encoding the heavy chain of an anti-CCL2 antibody has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: l or SEQ ID NO:2 as described herein.
  • an mRNA encoding the heavy chain of an anti-CCL2 antibody has a nucleotide sequence encoding an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to SEQ ID NO: l as described herein.
  • an mRNA encoding the light chain of an anti-CCL2 antibody has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:3 or SEQ ID NO:4 as described herein.
  • an mRNA encoding the light chain of an anti-CCL2 antibody has a nucleotide sequence encoding an amino acid sequence at least 50%>, 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to SEQ ID NO: 3 as described herein.
  • mRNA provided herein contains one or more modified nucleotides such as those described herein.
  • an mRNA encoding the heavy chain or light chain of an anti-CCL2 antibody may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%o, at least 90%>, or at least 95% of modified nucleotides of all modifiable nucleotides of the sequence.
  • Heavy chain anti-VEGF HC-aVEGF mRNA without 5 ' and 3 ' UTR (SEQ ID NO: 5):
  • Heavy chain anti-VEGF HC-aVEGF mRNA with 5 ' and 3 ' UTR (SEQ ID NO:6):
  • HC-aVEGF Light chain anti-VEGF mRNA without 5 ' and 3 ' UTR
  • HC-aVEGF Light chain anti-VEGF
  • the present invention also provides m NAs encoding a heavy chain and light chain of an anti-VEGF antibody substantially identical or similar to the sequences described herein.
  • an mRNA encoding the heavy chain of an anti- VEGF antibody has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO: 5 or SEQ ID NO: 6 as described herein.
  • an mRNA encoding the heavy chain of an anti- VEGF antibody has a nucleotide sequence encoding an amino acid sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to SEQ ID NO:5 as described herein.
  • an mRNA encoding the light chain of an anti-VEGF antibody has a nucleotide sequence at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to SEQ ID NO:7 or SEQ ID NO:8 as described herein.
  • an mRNA encoding the light chain of an anti- VEGF antibody has a nucleotide sequence encoding an amino acid sequence at least 50%>, 55%, 60%>, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical or homologous to SEQ ID NO: 7 as described herein.
  • mRNA provided herein contains one or more modified nucleotides such as those described herein.
  • an mRNA encoding the heavy chain or light chain of an anti-VEGF antibody may contain at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%o, at least 90%>, or at least 95% of modified nucleotides of all modifiable nucleotides of the sequence.
  • identity refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules ⁇ e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes ⁇ e.g. , gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%>, at least 40%>, at least 50%>, at least 60%>, at least 70%>, at least 80%>, at least 90%), at least 95%, or substantially 100%) of the length of the reference sequence.
  • the nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can,
  • antibody encoding mR As (e.g., heavy chain and light chain encoding mRNAs) described herein may be delivered as naked RNA
  • delivery vehicle delivery vehicle
  • transfer vehicle transfer vehicle
  • mRNAs encoding a heavy chain and a light chain of an antibody may be delivered via a single delivery vehicle. In some embodiments, mRNAs encoding a heavy chain and a light chain of an antibody may be delivered via separate delivery vehicles. For example, mRNAs encoding a heavy chain and a light chain of an antibody may be packaged separately but delivered simultaneously. Alternatively, mRNAs encoding a heavy chain and a light chain of an antibody may be packaged separately and delivered sequentially.
  • suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate
  • PEI polyethyleneimine
  • nanoparticulates silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder formulations, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags.
  • a suitable delivery vehicle is a liposomal delivery vehicle, e.g. a lipid nanoparticle.
  • liposomal delivery vehicles e.g. lipid nanoparticles
  • lipid nanoparticles are usually characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers.
  • Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends BiotechnoL, 16: 307-321, 1998).
  • Bilayer membranes of the liposomes can also be formed by amphophilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.).
  • a liposomal delivery vehicle typically serves to transport a desired mRNA to a target cell or tissue.
  • the process of incorporation of a desired mRNA into a liposome is often referred to as "loading". Exemplary methods are described in Lasic, et al, FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference.
  • the liposome -incorporated nucleic acids may be completely or partially located in the interior space of the liposome, within the bilayer membrane of the liposome, or associated with the exterior surface of the liposome membrane.
  • the incorporation of a nucleic acid into liposomes is also referred to herein as "encapsulation" wherein the nucleic acid is entirely contained within the interior space of the liposome.
  • the purpose of incorporating a mRNA into a transfer vehicle, such as a liposome is often to protect the nucleic acid from an environment which may contain enzymes or chemicals that degrade nucleic acids and/or systems or receptors that cause the rapid excretion of the nucleic acids. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the mRNA contained therein and/or facilitate the delivery of mRNA to the target cell or tissue.
  • a suitable delivery vehicle is formulated as a lipid nanoparticle.
  • lipid nanoparticle refers to a delivery vehicle comprising one or more lipids (e.g., cationic lipids, non-cationic lipids, cholesterol-based lipids, and PEG-modified lipids).
  • the contemplated lipid nanoparticles may be prepared by including multi-component lipid mixtures of varying ratios employing one or more cationic lipids, non- cationic lipids, cholesterol-based lipids, and PEG-modified lipids.
  • suitable lipids include, for example, the phosphatidyl compounds (e.g., phosphatidylglycerol,
  • phosphatidylcholine phosphatidylcholine
  • phosphatidylserine phosphatidylethanolamine
  • sphingolipids cerebrosides, and gangliosides.
  • the carrier is formulated using a polymer as a carrier, alone or in combination with other carriers.
  • Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI).
  • PEI polyethylenimine
  • it may be branched PEI of a molecular weight ranging from 10 to 40 kDA, e.g., 25 kDa branched PEI (Sigma #408727).
  • a suitable delivery vehicle contains a cationic lipid.
  • cationic lipid refers to any of a number of lipid species that have a net positive charge at a selected pH, such as physiological pH.
  • Several cationic lipids have been described in the literature, many of which are commercially available.
  • Particularly suitable cationic lipids for use in the compositions and methods of the invention include those described in international patent publications WO 2010/053572 (and particularly, CI 2-200 described at paragraph [00225]) and WO 2012/170930, both of which are incorporated herein by reference.
  • compositions and methods of the invention employ a lipid nanoparticles comprising an ionizable cationic lipid described in U.S. provisional patent application 61/617,468, filed March 29, 2012 (incorporated herein by reference), such as, e.g, (15Z, 18Z)-N,N-dimethyl-6-(9Z, 12Z)-octadeca-9, 12-dien-l -yl)tetracosa- 15,18-dien- 1 -amine (HGT5000), ( 15Z, 18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1 -yl)tetracosa- 4,15,18-trien-l -amine (HGT5001), and (15Z,18Z)-N,N-dimethyl-6-((9Z, 12Z)-octadeca-9, 12- dien- 1 -yl)tetracosa-5
  • the cationic lipid N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride or "DOTMA" is used.
  • DOTMA can be formulated alone or can be combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine or "DOPE" or other cationic or non- cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells.
  • suitable cationic lipids include, for example, 5-carboxyspermylglycinedioctadecylamide or "DOGS,” 2,3-dioleyloxy-N- [2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium or "DOSPA" (Behr et al. Proc.
  • Contemplated cationic lipids also include l,2-distearyloxy-N,N-dimethyl- 3-aminopropane or "DSDMA", l,2-dioleyloxy-N,N-dimethyl-3-aminopropane or "DODMA", 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane or "DLinDMA", l,2-dilinolenyloxy-N,N- dimethyl-3-aminopropane or "DLenDMA", N-dioleyl-N,N-dimethylammonium chloride or "DODAC", N,N-distearyl-N,N-dimethylarnrnonium bromide or "DDAB", N-(l,2- dimyristyloxyprop-3-yl)-N,N-dimethyl-N -hydroxy ethyl ammonium bromide or "DMRIE", 3- dimethylamino
  • one or more of the cationic lipids present in such a composition comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.
  • one or more of the cationic lipids present in such a composition are chosen from XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane), MC3 (((6Z,9Z,28Z,3 lZ)-heptatriaconta-6,9,28,3 l-tetraen-19-yl 4-(dimethylamino)butanoate), ALNY-100 ((3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH- cyclopenta[d] [1 ,3]dioxol-5-amine)), NC98-5 (4,7,13-tris(3-oxo-3-(undecylamino)propyl)- N 1 ,N 16
  • one or more of the cationic lipids present in such a composition is a cationic lipid described in WO 2013063468 and in U.S. provisional application serial number 61/894,299, entitled “Lipid Formulations for Delivery of Messernger RNA” filed on October 22, 2013, both of which are incorporated by reference herein.
  • a cationic lipid comprises a compound of formula I-cl-a:
  • each R 2 independently is hydrogen or Ci_ 3 alkyl; each q independently is 2 to 6; each R independently is hydrogen or Ci_ 3 alkyl; and each R L independently is C 8-12 alkyl.
  • each R 2 independently is hydrogen, methyl or ethyl. In some embodiments, each R 2 independently is hydrogen or methyl. In some embodiments, each R 2 is hydrogen. [0101] In some embodiments, each q independently is 3 to 6. In some embodiments, each q independently is 3 to 5. In some embodiments, each q is 4.
  • each R' independently is hydrogen, methyl or ethyl. In some embodiments, each R independently is hydrogen or methyl. In some embodiments, each R independently is hydrogen.
  • each R L independently is C 8-12 alkyl. In some embodiments, each R L independently is C 8-12 alkyl. In some
  • each R L independently is «-C 8-12 alkyl. In some embodiments, each R L independently is C9_n alkyl. In some embodiments, each R L independently is n-Cg-n alkyl. In some embodiments, each R L independently is C 10 alkyl. In some embodiments, each R L independently is n-C 10 alkyl.
  • each R 2 independently is hydrogen or methyl; each q independently is 3 to 5; each R independently is hydrogen or methyl; and each R L independently is C 8 -i2 alkyl.
  • each R 2 is hydrogen; each q independently is 3 to 5; each
  • R is hydrogen; and each R L independently is C 8-12 alkyl.
  • each R 2 is hydrogen; each q is 4; each R' is hydrogen; and each R L independently is C 8-12 alkyl.
  • a cationic lipid comprises a compound of formula I-g:
  • each R L independently is C 8-12 alkyl. In some embodiments, each R L independently is «-C 8-12 alkyl. In some embodiments, each R L independently is C9_n alkyl. In some embodiments, each R L independently is n-Cg-n alkyl. In some embodiments, each R L independently is Cio alkyl. In some embodiments, each R L is n-C 10 alkyl.
  • compositions include a cationic lipid cK -
  • a suitable delivery vehicle contains one or more non- cationic lipids,
  • a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the composition is formulated and/or administered.
  • Such exemplary non-cationic or neutral lipids can be chosen from DSPC (1 ,2- distearoyl-sn-glycero-3-phosphocholine), DPPC (1 ,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (l ,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (l ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine), DMPE (l ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (l ,2-dioleoyl-sn-glycero-3-phospho-(l'-rac-glycerol)), and cholesterol.
  • DSPC 1,2- distearoyl-sn-glycero-3-phosphocholine
  • DPPC 1,2-dipalmitoyl-sn-glycero-3-phosphocholine
  • DOPE l ,2-d
  • cholesterol-based cationic lipids are also contemplated by the present invention.
  • Such cholesterol-based cationic lipids can be used, either alone or in combination with other cationic or non-cationic lipids.
  • Suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or ICE.
  • suitable lipid nanoparticles comprising one or more cleavable lipids, such as, for example, one or more cationic lipids or compounds that comprise a cleavable disulfide (S-S) functional group (e.g., HGT4001, HGT4002, HGT4003, HGT4004 and HGT4005), as further described in U.S. Provisional Application No: 61/494,745, the entire teachings of which are incorporated herein by reference in their entirety.
  • S-S cleavable disulfide
  • LIPOFECTIN DOTMA:DOPE
  • LIPOFECTA INE DOSPA:DOPE
  • LIPOFECTAMINE2000 LIPOFECTAMINE2000.
  • FUGENE FUGENE
  • TRANSFECTAM DOGS
  • EFFECTENE EFFECTENE
  • the cationic lipid may comprise a molar ratio of about 1% to about 90%, about 2% to about 70%, about 5% to about 50%, about 10% to about 40% of the total lipid present in the transfer vehicle, or preferably about 20% to about 70% of the total lipid present in the transfer vehicle.
  • PEG polyethylene glycol
  • PEG-CER derivatized cerarmides
  • N-Octanoyl-Sphingosine-1- succinyl(Methoxy Polyethylene Glycol)-2000
  • C8 PEG-2000 ceramide C8 PEG-2000 ceramide
  • Contemplated PEG-modified lipids include, but is not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length.
  • the addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid composition to the target cell, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the formulation in vivo (see U.S. Pat. No. 5,885,613).
  • Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18).
  • the PEG-modified phospholipid and derivitized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle.
  • non-cationic lipid refers to any neutral, zwitterionic or anionic lipid.
  • anionic lipid refers to any of a number of lipid species that carry a net negative charge at a selected H, such as physiological pH.
  • Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG),
  • dipalmitoylphosphatidylglycerol DPPG
  • dioleoylphosphatidylethanolamine DOPE
  • palmitoyloleoylphosphatidylcholine POPC
  • palmitoyloleoyl-phosphatidylethanolamine POPE
  • dipalmitoyl phosphatidyl ethanolamine DPPE
  • dimyristoylphosphoethanolamine DMPE
  • distearoyl-phosphatidyl-ethanolamine DSPE
  • 16-O-monomethyl PE 16-O-dimethyl PE
  • 18-1 -trans PE l-stearoyl-2-oleoyl-phosphatidyethanolamine
  • SOPE l-stearoyl-2-oleoyl-phosphatidyethanolamine
  • non-cationic lipids may be used alone, but are preferably used in combination with other excipients, for example, cationic lipids.
  • the non-cationic lipid may comprise a molar ratio of 5% to about 90%, or preferably about 10 % to about 70% of the total lipid present in the transfer vehicle.
  • a suitable transfer vehicle e.g., a lipid nanoparticle
  • a transfer vehicle may be prepared using CI 2-200, DOPE, chol, DMG-PEG2K at a molar ratio of
  • HGT5000 DOPE, chol, DMG-PEG2K at a molar ratio of 40:20:35:5, or HGT5001, DOPE, chol,
  • DMG-PEG2K at a molar ratio of 40:20:35:5.
  • the selection of cationic lipids, non-cationic lipids and/or PEG-modified lipids which comprise the lipid nanoparticle, as well as the relative molar ratio of such lipids to each other, is based upon the characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the mRNA to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.
  • the percentage of cationic lipid in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, greater than 40%, greater than 50%, greater than 60%, or greater than 70%.
  • the percentage of non-cationic lipid in the lipid nanoparticle may be greater than 5%, greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • the percentage of cholesterol in the lipid nanoparticle may be greater than 10%, greater than 20%, greater than 30%, or greater than 40%.
  • the percentage of PEG- modified lipid in the lipid nanoparticle may be greater than 1%, greater than 2%, greater than 5%, greater than 10%, or greater than 20%.
  • suitable lipid nanoparticles of the invention comprise at least one of the following cationic lipids: CI 2-200, DLin-KC2-DMA, DODAP, HGT4003, ICE, HGT5000, or HGT5001.
  • suitable transfer vehicle comprises cholesterol and/or a PEG-modified lipid.
  • suitable transfer vehicles comprises DMG- PEG2K.
  • suitable transfer vehicle comprises one of the following lipid combinations: C 12-200, DOPE, cholesterol, DMG-PEG2K; DODAP, DOPE, cholesterol, DMG- PEG2K; HGT5000, DOPE, cholesterol, DMG-PEG2K; HGT5001, DOPE, cholesterol, DMG- PEG2K; XTC, DSPC, cholesterol, PEG-DMG; MC3, DSPC, cholesterol, PEG-DMG; and ALNY-100, DSPC, cholesterol, PEG-DSG.
  • the liposomal transfer vehicles for use in the compositions of the invention can be prepared by various techniques which are presently known in the art.
  • Multilamellar vesicles may be prepared conventional techniques, for example, by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then added to the vessel with a vortexing motion which results in the formation of MLVs.
  • Uni-lamellar vesicles UUV
  • UUV can then be formed by homogenization, sonication or extrusion of the multi-lamellar vesicles.
  • compositions of the present invention comprise a transfer vehicle wherein the mRNA is associated on both the surface of the transfer vehicle and encapsulated within the same transfer vehicle.
  • cationic liposomal transfer vehicles may associate with the mRNA through electrostatic interactions.
  • cationic liposomal transfer vehicles may associate with the mRNA through electrostatic interactions.
  • Suitable liposomal delivery vehicles according to the present invention may be made in various sizes. Selection of an appropriate size may take into consideration the site of the target cell or tissue and to some extent the application for which the liposome is being made. In some embodiments, an appropriate size of liposomal delivery vehicles is selected to facilitate systemic distribution of antibody encoded by the mRNA. In some embodiments, it may be desirable to limit transfection of the mRNA to certain cells or tissues.
  • a liposomal transfer vehicle may be sized such that its dimensions are smaller than the fenestrations of the endothelial layer lining hepatic sinusoids in the liver; accordingly the liposomal transfer vehicle can readily penetrate such endothelial fenestrations to reach the target hepatocytes.
  • a liposomal transfer vehicle may be sized such that the dimensions of the liposome are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues.
  • a liposomal transfer vehicle may be sized such that its dimensions are larger than the fenestrations of the endothelial layer lining hepatic sinusoids to thereby limit distribution of the liposomal transfer vehicle to hepatocytes.
  • a suitable liposomal delivery vehicle has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 nm, or 50 nm). In some embodiments, a suitable liposomal delivery vehicle has a size ranging from about 250 - 10 nm (e.g., ranging from about 225 - 10 nm, 200 - 10 nm, 175 - 10 nm, 150 - 10 nm, 125 - 10 nm, 100 - 10 nm, 75 - 10 nm, or 50 - 10 nm). In some embodiments, a suitable liposomal delivery vehicle has a size no greater than about 250 nm (e.g., no greater than about 225 nm, 200 nm, 175 nm, 150 nm, 125 nm, 100 nm, 75 n
  • a suitable liposomal delivery vehicle has a size ranging from about 250 - 100 nm (e.g., ranging from about 225 - 100 nm, 200 - 100 nm, 175 - 100 nm, 150 - 100 nm). In some embodiments, a suitable liposomal delivery vehicle has a size ranging from about 100 - 10 nm (e.g., ranging from about 90 - 10 nm, 80 - 10 nm, 70 - 10 nm, 60 - 10 nm, or 50 - 10 nm). [0123] A variety of alternative methods known in the art are available for sizing of a population of liposomal transfer vehicles.
  • Average liposome diameter may be reduced by sonication of formed liposomes. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient liposome synthesis.
  • antibody encoding mRNAs e.g., heavy chain and light chain encoding mRNAs
  • mRNAs e.g., heavy chain and light chain encoding mRNAs
  • described herein may be delivered, with or without delivery vehicles, to a subject in need of delivery such that a fully assembled desired antibody is expressed in vivo.
  • a desired antibody encoded by mRNAs is expressed systemically in the subject. This can be achieved by secreting fully assembled antibodies from a cell or tissue in which the antibody is initially expressed into the circulation system of the subject.
  • compositions of the invention containing antibody encoding mRNAs and lipososmal vehicles distribute into the cells of the liver to facilitate the delivery and the subsequent expression of the mRNA comprised therein by the cells of the liver (e.g., hepatocytes).
  • the targeted hepatocytes may function as a biological "reservoir” or “depot” capable of producing, and excreting a fully assembled desired antibody, resulting in systemic distribution of the antibody.
  • cells other than hepatocytes can serve as a depot location for protein production.
  • sustained production and secretion of fully assembled antibodies from the reservoir or depot cells results in effective systemic distribution.
  • systemic expression of a desired antibody encoded mRNAs in the patient serum (i.e., blood) is detectable for more than 1 hour, more than 4 hours, more than 6 hours, more than 12 hours, more than 18 hours, more than 24 hours, more than 30 hours, more than 36 hours, more than 42 hours, more than 48 hours, more than 54 hours, more than 60 hours, more than 66 hours, more than 72 hours, more than 96 hours, more than 120 hours, more than 144 hours, more than 168 hours, more than 2 weeks, more than 3 weeks, more than 1 month or more than 2 months after administration.
  • the serum concentration of the antibody encoded by mRNAs reaches a peak level about 6 hours, 12 hours, 18 hours, 24 hours, 30 hours, 36 hours, 42 hours, 48 hours, 54 hours, 60 hours, 66 hours, 72 hours, 78 hours, 84 hours, 90 hours, or 96 hours after administration.
  • sustained circulation of the desired antibody encoded by mRNAs are observed.
  • the systemic expression of the antibody encoded by mRNAs in the patient serum i.e., blood
  • mRNAs encoding heavy chain and light chain of an antibody may be delivered to target cells or tissues for intracellular expression or local distribution of the antibody.
  • target cells or tissues typically, local distribution results when a fully assembled antibody is produced and secreted from a target cell to the surrounding extracellular fluid without entering the circulation system, such as blood stream.
  • target cell or “target tissue” refers to a cell or tissue to which antibody encoding mRNA(s) is to be directed or targeted. For example, where it is desired to deliver an mRNA to a hepatocyte, the hepatocyte represents the target cell.
  • Antibody encoding mRNAs may be delivered to a variety of target cells or tissues including, but not limited to, hepatocytes, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells, bone cells, stem cells, mesenchymal cells, neural cells (e.g., meninges, astrocytes, motor neurons, cells of the dorsal root ganglia and anterior horn motor neurons), photoreceptor cells (e.g., rods and cones), retinal pigmented epithelial cells, secretory cells, cardiac cells, adipocytes, vascular smooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells, synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes and tumor cells.
  • target cells or tissues including, but not limited to, hepatocytes, epit
  • Delivery of mRNAs to target cells and tissues may be accomplished by both passive and active targeting means.
  • passive targeting exploits the natural distributions patterns of a transfer vehicle in vivo without relying upon the use of additional excipients or means to enhance recognition of the transfer vehicle by target cells.
  • transfer vehicles which are subject to phagocytosis by the cells of the reticulo-endothelial system are likely to accumulate in the liver or spleen, and accordingly may provide means to passively direct the delivery of the compositions to such target cells.
  • mRNAs to target cells and tissues may be accomplished by active targeting, which involves the use of additional excipients, referred to herein as
  • targeting ligands that may be bound (either covalently or non-covalently) to the transfer vehicle to encourage localization of such transfer vehicle at certain target cells or target tissues.
  • targeting may be mediated by the inclusion of one or more endogenous targeting ligands (e.g., apolipoprotein E) in or on the transfer vehicle to encourage distribution to the target cells or tissues.
  • endogenous targeting ligands e.g., apolipoprotein E
  • Recognition of the targeting ligand by the target tissues actively facilitates tissue distribution and cellular uptake of the transfer vehicle and/or its contents in the target cells and tissues (e.g., the inclusion of an apolipoprotein-E targeting ligand in or on the transfer vehicle encourages recognition and binding of the transfer vehicle to endogenous low density lipoprotein receptors expressed by hepatocytes).
  • the composition can comprise a ligand capable of enhancing affinity of the composition to the target cell.
  • Targeting ligands may be linked to the outer bilayer of the lipid particle during formulation or post- formulation. These methods are well known in the art.
  • some lipid particle formulations may employ fusogenic polymers such as PEAA, Hemagglutinin , other lipopeptides (see U.S. Patent
  • compositions of the present invention demonstrate improved transfection efficacies, and/or demonstrate enhanced selectivity towards target cells or tissues of interest.
  • Contemplated therefore are compositions which comprise one or more ligands (e.g., peptides, aptamers, oligonucleotides, a vitamin or other molecules) that are capable of enhancing the affinity of the compositions and their nucleic acid contents for the target cells or tissues.
  • ligands may optionally be bound or linked to the surface of the transfer vehicle.
  • the targeting ligand may span the surface of a transfer vehicle or be encapsulated within the transfer vehicle.
  • Suitable ligands and are selected based upon their physical, chemical or biological properties (e.g., selective affinity and/or recognition of target cell surface markers or features) Cell-specific target sites and their corresponding targeting ligand can vary widely. Suitable targeting ligands are selected such that the unique characteristics of a target cell are exploited, thus allowing the composition to discriminate between target and non-target cells.
  • compositions of the invention may include surface markers (e.g., apolipoprotein-B or apolipoprotein-E) that selectively enhance recognition of, or affinity to hepatocytes (e.g., by receptor-mediated recognition of and binding to such surface markers).
  • surface markers e.g., apolipoprotein-B or apolipoprotein-E
  • galactose as a targeting ligand would be expected to direct the compositions of the present invention to parenchymal hepatocytes
  • mannose containing sugar residues as a targeting ligand would be expected to direct the compositions of the present invention to liver endothelial cells (e.g., mannose containing sugar residues that may bind preferentially to the asialoglycoprotein receptor or mannose receptor present in hepatocytes).
  • targeting ligands that have been conjugated to moieties present in the transfer vehicle (e.g., a lipid nanoparticle) therefore facilitate recognition and uptake of the compositions of the present invention in target cells and tissues.
  • suitable targeting ligands include one or more peptides, proteins, aptamers, vitamins and oligonucleotides.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, to which the mR As and compositions of the present invention are administered.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • antibody encoding mR As e.g., heavy chain and light chain encoding mRNAs
  • delivery vehicles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients.
  • Antibody encoding mRNAs and compositions containing the same may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.
  • the "effective amount" for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical and medical arts.
  • the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art.
  • a suitable amount and dosing regimen is one that causes at least transient antibody production.
  • Suitable routes of administration include, for example, oral, rectal, vaginal, transmucosal, pulmonary including intratracheal or inhaled, or intestinal administration;
  • parenteral delivery including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections.
  • mRNAs and compositions of the invention may be administered in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a targeted tissue, preferably in a sustained release formulation. Local delivery can be affected in various ways, depending on the tissue to be targeted.
  • compositions of the present invention can be inhaled (for nasal, tracheal, or bronchial delivery); compositions of the present invention can be injected into the site of injury, disease manifestation, or pain, for example; compositions can be provided in lozenges for oral, tracheal, or esophageal application; can be supplied in liquid, tablet or capsule form for administration to the stomach or intestines, can be supplied in suppository form for rectal or vaginal application; or can even be delivered to the eye by use of creams, drops, or even injection.
  • Formulations containing compositions of the present invention complexed with therapeutic molecules or ligands can even be surgically administered, for example in association with a polymer or other structure or substance that can allow the compositions to diffuse from the site of implantation to surrounding cells. Alternatively, they can be applied surgically without the use of polymers or supports.
  • compositions of the invention are formulated such that they are suitable for extended-release of the mR A contained therein.
  • extended-release compositions may be conveniently administered to a subject at extended dosing intervals.
  • the compositions of the present invention are administered to a subject twice day, daily or every other day.
  • the compositions of the present invention are administered to a subject twice a week, once a week, every ten days, every two weeks, every three weeks, or more preferably every four weeks, once a month, every six weeks, every eight weeks, every other month, every three months, every four months, every six months, every eight months, every nine months or annually.
  • compositions and liposomal vehicles which are formulated for depot administration (e.g., intramuscularly, subcutaneously, intravitreally) to either deliver or release a mRNA over extended periods of time.
  • depot administration e.g., intramuscularly, subcutaneously, intravitreally
  • the extended-release means employed are combined with modifications made to the mRNA to enhance stability.
  • lyophilized pharmaceutical compositions comprising one or more of the liposomal nanoparticles disclosed herein and related methods for the use of such lyophilized compositions as disclosed for example, in United States Provisional Application No. 61/494,882, filed June 8, 2011, the teachings of which are incorporated herein by reference in their entirety.
  • lyophilized pharmaceutical compositions according to the invention may be reconstituted prior to administration or can be reconstituted in vivo.
  • a lyophilized pharmaceutical composition can be formulated in an appropriate dosage form (e.g., an intradermal dosage form such as a disk, rod or membrane) and administered such that the dosage form is rehydrated over time in vivo by the individual's bodily fluids.
  • antibody encoding mRNAs may be used to produce antibodies in vitro.
  • cells may be transfected by antibody encoding mRNAs (e.g., heavy chain and light chain encoding mRNAs) and cultured under cell culture conditions that allow the production of the antibody by the cells.
  • the antibody is expressed intracellularly.
  • the antibody is secreted by the cells such that the antibody may be harvested from the supernatant.
  • mammalian cells are used in accordance with the present invention.
  • mammalian cells include BALB/c mouse myeloma line (NSO/1, ECACC No: 85110503); human retinoblasts (PER.C6, CruCell, Leiden, The
  • human embryonic kidney line (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al, J. Gen Virol, 36:59,1977); human fibrosarcoma cell line (e.g., HT1080); baby hamster kidney cells (BHK21, ATCC CCL 10); Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol.
  • HEK293 or 293 cells subcloned for growth in suspension culture Graham et al, J. Gen Virol, 36:59,1977
  • human fibrosarcoma cell line e.g., HT1080
  • baby hamster kidney cells BHK21, ATCC CCL 10
  • Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA,
  • monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al, Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2).
  • Standard cell culture media and conditions may be used to cultivate transfected cells and produce desired antibodies encoded by mRNAs.
  • Heavy chain anti-chemokine (C-C motif) ligand 2 (HC-aCCL2, SEQ ID NO: 1) and light chain anti-CCL2 (LC-aCCL2, SEQ ID NO: 2) were synthesized by in vitro transcription from a plasmid DNA template encoding the gene, which was followed by the addition of a 5' cap structure (Capl) according to known methods ⁇ see Fechter, P.; Brownlee, G.G. "Recognition of mRNA cap structures by viral and cellular proteins" J. Gen. Virology 2005, 86, 1239-1249) and a 3' poly(A) tail of approximately 200 nucleotides in length as determined by gel electrophoresis.
  • the sequences for HC-aCCL2 and LC-aCCL2 were as shown below, and 5 ' and 3 ' untranslated regions present in each mRNA product are represented as X and Y, respectively, and defined below:
  • Heavy chain anti-CCL2 (HC-aCCL2) mRNA:
  • LC-aCCL2 Light chain anti-CCL2
  • the lipid formulations used for transfection in the examples herein consisted of one or more lipids or a multi-component lipid mixture of varying ratios employing one or more cationic lipids, helper lipids and PEGylated lipids designed to encapsulate various nucleic acid- based materials.
  • Cationic lipids can include (but not exclusively) DOTAP (l,2-dioleyl-3- trimethylammonium propane), DODAP (l ,2-dioleyl-3-dimethylammonium propane) , DOTMA (l ,2-di-0-octadecenyl-3-trimethylammonium propane), DLinDMA (see Heyes, J.; Palmer, L.; Bremner, K.; MacLachlan, I. "Cationic lipid saturation influences intracellular delivery of encapsulated nucleic acids" J. Contr. Rel. 2005, 107, 276-287), DLin-KC2-DMA (see Semple, S.C. et al.
  • Helper lipids can include (but not exclusively) DSPC (1 ,2-distearoyl- sn-glycero-3-phosphocholine), DPPC (l ,2-dipalmitoyl-sn-glycero-3-phosphocholine), DOPE (1 ,2-dioleyl-sn-glycero-3-phosphoethanolamine), DPPE (1 ,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine), DMPE (l ,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (2- dioleoyl-sn-glycero-3-phospho-(l ac-glycerol)), cholesterol, etc.
  • the PEGylated lipids can include (but not exclusively) a poly(ethylene) glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C 6 -C2o length.
  • DOPE, cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.
  • an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of HC- aCCL2 mRNA and LC-aCCL2 mRNA (1 : 1 w wt) mRNA was prepared by addition of 500 microgram of each construct from a 1 mg/mL stock.
  • the lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol.
  • the resulting nanoparticle suspension was filtered, diafiltrated with lx PBS (pH 7.4), concentrated and stored at 2-8°C.
  • Final concentration 1.35 mg/mL aCCL2 mRNA (encapsulated).
  • HCL2 cells were fractionated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane using a transfer apparatus according to the manufacturer's instructions (Invitrogen). After incubation with 5% non-fat dry milk in TBST (lOmM Tris, pH 8.0, 150mM NaCl, 0.5% Tween 20) for Ice, the membrane was washed three times with PBST and incubated with goat anti mouse IgGl (Invitrogen A 10538) for HIV at RT. Membranes were washed three times in PBST and incubated with 1 :5000 dilution of horseradish peroxidase conjugated anti- mouse secondary antibody (Promega W4021) for Jackpot at RT. Blots were washed in PBST three more times and developed with the ECL system (Amersham Bioscience) according to manufacturer's instructions.
  • Example 1 Subsequently, in accordance with Example 2A and B, HC-aCCL2 and LC-aCCL2 mRNA was transfected into either HCL1 (i.e., human cell line 1) cells or HCL2 cells in various rations (w wt) according to known methods. [0146] The results of these studies in HCLl cells and HCL2 cells are demonstrated in
  • FIGS 1 and 2 respectively.
  • Various mixtures of a-CCL2 (chemokine (C-C motif) ligand 2) heavy chain and light chain mR A constructs were mixed (wtwt) and transfected into either HCLl cells ( Figure 1) or HCL2 cells ( Figure 2).
  • Cell supematants were harvested at select time points post-transfection and analyzed for the presence of anti-mouse IgG using ELISA-based methods as described above in Example 3A.
  • Example 5 In Vivo Analysis of aCCL2 Antibody Produced from Intravenously Administered mRNA-Loaded Nanoparticles
  • a-CCL2 heavy chain and light chain mRNA constructs (HC-aCCL2:LC-aCCL2 mRNA, 1 : 1 (wtwt)) were encapsulated in cationic lipid nanoparticles as described in Example 2A and delivered to mice as a single bolus, intravenous injection.
  • a-CCL2 heavy chain and light chain mRNA constructs (HC-aCCL2:LC-aCCL2 mRNA, 1 : 1 (wtwt)) were encapsulated in cationic lipid nanoparticles as described in Example 2A and delivered to mice as a single bolus, intravenous injection.
  • male CD-I mice of approximately 6-8 weeks of age at the beginning of each experiment were used.
  • Samples of encapsulated HC-aCCL2 mR A and LC-aCCL2 mR A (1 : 1 w wt) were introduced by a single bolus tail-vein injection of an equivalent total dose of 30 micrograms. Mice were sacrificed and perfused with saline at the designated time points.
  • liver and spleen of each mouse was harvested, apportioned into three parts, and stored in either 10% neutral buffered formalin or snap-frozen and stored at -80°C for analysis.
  • Immuno Plate were coated with 100ml of lmg/ml of MCP-1 recombinant rabbit purified monoclonal antibody in carbonate buffer, pH 9.6 and incubated lhr at 37°C. After washing 3x with wash buffer (IxPBS, 0.05%> Tween 20), the wells were blocked with 320 ml blocking buffer (IxPBS, 0.05% Tween 20, 2% BSA) for lhr at 37°C. Approximately lOOng/ml MCP-1 human or mouse recombinant protein was added to each well and incubated for lhr at 37°C.
  • Heavy chain anti-VEGF (HC-aVEGF) mRNA Heavy chain anti-VEGF (HC-aVEGF) mRNA:
  • LC-aVEGF wiRNA Light chain anti-VEGF wiRNA
  • a-VEGF heavy chain and light chain mR A constructs (HC-a-VEGF:LC-a-
  • VEGF mRNA, 1 :1 (w wt)) were encapsulated in cationic lipid nanoparticles as described below:
  • DOPE, cholesterol and DMG-PEG2K were mixed and diluted with ethanol to 3 mL final volume.
  • an aqueous buffered solution (10 mM citrate/150 mM NaCl, pH 4.5) of HC- aCVEGF mRNA and LC-aVEGF mR A (1 : 1 wt:wt) mRNA was prepared by addition of 500 microgram of each construct from a 1 mg/mL stock.
  • the lipid solution was injected rapidly into the aqueous mRNA solution and shaken to yield a final suspension in 20% ethanol.
  • the resulting nanoparticle suspension was filtered, diafiltrated with lx PBS (pH 7.4), concentrated and stored at 2-8°C.
  • Final concentration 0.20 mg/mL aVEGF mRNA (encapsulated).
  • HC-a-VEGF and LC-a-VEGF mRNA loaded lipid nanoparticles were delivered to wild type mice either by a single intravenous tail vein injection or subcutaneous injection at a dosage of 1.0 mg/kg and the production of anti-VEGF antibody was monitored over time in serum via ELISA.
  • mice Male CD-I mice of approximately 6-8 weeks of age at the beginning of each experiment were used. Samples of encapsulated HC-a-VEGF mRNA and LC-a-VEGF mRNA (1 : 1 w wt) were introduced by a single bolus tail-vein injection of an equivalent total dose of 1.0 mg/kg (-30 micrograms). Mice were sacrificed and perfused with saline at the designated time points (0.50 hour, 3 hours, six hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks post-administration.
  • Immuno Plate were coated with 100 microliters of 0.50 microgram/mL/well of recombinant human VEGF protein(Invitrogen #PHC9391) in coating buffer (50mM NaHC0 3 , pH9.6). After washing 3x with wash buffer, wells were blocked using a blocking buffer (IX DPBS, 2% BSA, 0.05% Tween-20) for one hour at 37°C. Upon further washing as described above, mouse serum collected from injected mice were added to each well and rabbit anti-human IgG Fc HRP (Pierce
  • Figure 5 depicts exemplary results illustrating a-VEGF antibody detected in serum of wild type mice after single dose of HC-a-VEGF mRNA and LC-a-VEGF mRNA loaded nanoparticles.
  • a significant increase in the desired, exogenous a-VEGF mRNA derived antibody can be observed within six hours post-administration with a peak after 24 hours and continued out to 2 weeks after a single dose of a-VEGF mRNA.
  • Figure 6 depicts the same exemplary results as Figure 5, but plotted by specific mouse number.
  • Figure 7 shows a comparison of the levels of a-VEGF antibody present in the serum of mice injected either intravenously or subcutaneously after 24 hours.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014152774A1 (en) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Methods and compositions for delivering mrna coded antibodies
US11421038B2 (en) 2007-01-09 2022-08-23 Curevac Ag RNA-coded antibody

Families Citing this family (82)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
PT2506857T (pt) 2009-12-01 2018-05-14 Translate Bio Inc Entrega de arnm para o acréscimo de proteínas e enzimas em doenças genéticas humanas
US9238716B2 (en) 2011-03-28 2016-01-19 Massachusetts Institute Of Technology Conjugated lipomers and uses thereof
SI2717893T1 (sl) 2011-06-08 2019-10-30 Translate Bio Inc Sestavki lipidnih nanodelcev in postopki za dostavo mRNA
EP4074693A1 (de) 2011-06-08 2022-10-19 Translate Bio, Inc. Spaltbare lipide
EA032088B1 (ru) 2011-10-27 2019-04-30 Массачусетс Инститьют Оф Текнолоджи Аминокислотные производные, функционализованные на n-конце, способные образовывать микросферы, инкапсулирующие лекарственное средство
EP3536787A1 (de) 2012-06-08 2019-09-11 Translate Bio, Inc. Nukleaseresistente polynukleotide und verwendungen davon
KR102311614B1 (ko) 2013-03-14 2021-10-08 샤이어 휴먼 지네틱 테라피즈 인크. Cftr mrna 조성물 및 관련 방법 및 사용
AU2014236396A1 (en) 2013-03-14 2015-08-13 Shire Human Genetic Therapies, Inc. Methods for purification of messenger RNA
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
AU2014329452B2 (en) 2013-10-03 2019-06-20 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor
CN105658242A (zh) 2013-10-22 2016-06-08 夏尔人类遗传性治疗公司 用于苯丙酮尿症的mrna疗法
AU2014340092B2 (en) 2013-10-22 2019-09-19 Translate Bio, Inc. mRNA therapy for Argininosuccinate Synthetase Deficiency
CA2928078A1 (en) 2013-10-22 2015-04-30 Shire Human Genetic Therapies, Inc. Lipid formulations for delivery of messenger rna
EP3092250A4 (de) 2014-01-08 2017-05-24 Moderna Therapeutics, Inc. Polynukleotide zur in-vivo-erzeugung von antikörpern
EP3450553B1 (de) * 2014-03-24 2019-12-25 Translate Bio, Inc. Mrna-therapie zur behandlung von augenerkrankungen
SG11201608798YA (en) 2014-04-23 2016-11-29 Modernatx Inc Nucleic acid vaccines
CN110511927A (zh) 2014-04-25 2019-11-29 川斯勒佰尔公司 信使rna的纯化方法
MA40871A (fr) * 2014-10-29 2017-09-05 Novartis Ag Expression directe d'anticorps
MA56412A (fr) * 2014-12-05 2022-05-04 Translate Bio Inc Thérapie par l'arn messager pour le traitement des maladies articulaires
EP3900702A1 (de) * 2015-03-19 2021-10-27 Translate Bio, Inc. Mrna-therapie für morbus pompe
DK3294885T3 (da) 2015-05-08 2020-08-10 Curevac Real Estate Gmbh Fremgangsmåde til at fremstille rna
DE202016009003U1 (de) 2015-05-29 2021-05-28 Curevac Real Estate Gmbh Zusammensetzung umfassend in vitro transkribierte RNA erhältlich durch ein Verfahren zur Herstellung und Reinigung von RNA mit mindestens einem Schritt mit einer tangentialen Flussfiltration
CA2990172A1 (en) * 2015-06-19 2016-12-22 Massachusetts Institute Of Technology Alkenyl substituted 2,5-piperazinediones and their use in compositions for delivering an agent to a subject or cell
US11007260B2 (en) 2015-07-21 2021-05-18 Modernatx, Inc. Infectious disease vaccines
US11364292B2 (en) 2015-07-21 2022-06-21 Modernatx, Inc. CHIKV RNA vaccines
US20190008887A1 (en) * 2015-07-30 2019-01-10 ModernaTX Inc. Multimeric mrna
AU2016342048B2 (en) * 2015-10-22 2022-09-08 Modernatx, Inc. Broad spectrum influenza virus vaccine
AU2016342376A1 (en) * 2015-10-22 2018-06-07 Modernatx, Inc. Sexually transmitted disease vaccines
CN108472309A (zh) * 2015-10-22 2018-08-31 摩登纳特斯有限公司 用于水痘带状疱疹病毒（vzv）的核酸疫苗
JP2018536023A (ja) * 2015-10-22 2018-12-06 モデルナティーエックス， インコーポレイテッド 単純ヘルペスウイルスワクチン
SG11201803363YA (en) * 2015-10-22 2018-05-30 Modernatx Inc Respiratory syncytial virus vaccine
SI3718565T1 (sl) 2015-10-22 2022-08-31 Modernatx, Inc. Cepiva za respiratorni virus
CA3002922A1 (en) * 2015-10-22 2017-04-27 Modernatx, Inc. Human cytomegalovirus vaccine
WO2017070624A1 (en) 2015-10-22 2017-04-27 Modernatx, Inc. Tropical disease vaccines
WO2017099823A1 (en) 2015-12-10 2017-06-15 Modernatx, Inc. Compositions and methods for delivery of therapeutic agents
US20210206818A1 (en) 2016-01-22 2021-07-08 Modernatx, Inc. Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof
WO2017180917A2 (en) 2016-04-13 2017-10-19 Modernatx, Inc. Lipid compositions and their uses for intratumoral polynucleotide delivery
DK3458083T3 (da) 2016-05-18 2023-01-30 Modernatx Inc Polynukleotider, der koder for interleukin-12 (il12), og anvendelser heraf
BR112018073683A2 (pt) 2016-05-18 2019-02-26 Modernatx, Inc. polinucleotídeos codificadores de relaxina
WO2018075980A1 (en) 2016-10-21 2018-04-26 Modernatx, Inc. Human cytomegalovirus vaccine
WO2018107088A2 (en) 2016-12-08 2018-06-14 Modernatx, Inc. Respiratory virus nucleic acid vaccines
EP3582790A4 (de) 2017-02-16 2020-11-25 ModernaTX, Inc. Hochwirksame immunogene zusammensetzungen
AU2018224318A1 (en) 2017-02-27 2019-09-19 Translate Bio, Inc. Methods for purification of messenger RNA
HUE059025T2 (hu) 2017-02-27 2022-10-28 Translate Bio Inc Módszerek a hírvívõ RNS tisztítására
MA47603A (fr) 2017-02-27 2020-01-01 Translate Bio Inc Nouvel arnm cftr à codons optimisés
MA52262A (fr) 2017-03-15 2020-02-19 Modernatx Inc Vaccin à large spectre contre le virus de la grippe
MA47787A (fr) 2017-03-15 2020-01-22 Modernatx Inc Vaccin contre le virus respiratoire syncytial
WO2018170347A1 (en) 2017-03-17 2018-09-20 Modernatx, Inc. Zoonotic disease rna vaccines
IL270631B2 (en) 2017-05-16 2024-03-01 Translate Bio Inc Treatment of cystic fibrosis through the administration of mRNA with an optimal codon encoding ctfr
US20200131498A1 (en) 2017-06-14 2020-04-30 Modernatx, Inc. Polynucleotides encoding methylmalonyl-coa mutase
MA50253A (fr) 2017-09-14 2020-07-22 Modernatx Inc Vaccins à arn contre le virus zika
EP3746090A4 (de) 2018-01-29 2021-11-17 ModernaTX, Inc. Rsv-rns-impfstoffe
TW202019518A (zh) 2018-07-13 2020-06-01 丹麥商珍美寶股份有限公司 Ｃｄ３８抗體之變體及其用途
MA53123A (fr) 2018-07-13 2021-05-19 Genmab As Thérapie à médiation par trogocytose utilisant des anticorps cd38
CN112930396A (zh) 2018-08-24 2021-06-08 川斯勒佰尔公司 用于纯化信使rna的方法
CN113166783A (zh) 2018-10-09 2021-07-23 不列颠哥伦比亚大学 包含无有机溶剂和去污剂的转染活性囊泡的组合物和系统以及与之相关的方法
EP3877538A1 (de) 2018-11-08 2021-09-15 Translate Bio, Inc. Verfahren und zusammensetzungen zur aufreinigung von messenger-rna
US11351242B1 (en) 2019-02-12 2022-06-07 Modernatx, Inc. HMPV/hPIV3 mRNA vaccine composition
WO2021154763A1 (en) 2020-01-28 2021-08-05 Modernatx, Inc. Coronavirus rna vaccines
US20230346914A1 (en) 2020-02-07 2023-11-02 Modernatx, Inc. Sars-cov-2 mrna domain vaccines
WO2021222304A1 (en) 2020-04-27 2021-11-04 Modernatx, Inc. Sars-cov-2 rna vaccines
WO2021159130A2 (en) 2020-05-15 2021-08-12 Modernatx, Inc. Coronavirus rna vaccines and methods of use
CN111658782A (zh) * 2020-06-10 2020-09-15 深圳近邻生物科技有限公司 mRNA疫苗递送载体及制备方法、mRNA疫苗及制备方法
US11406703B2 (en) 2020-08-25 2022-08-09 Modernatx, Inc. Human cytomegalovirus vaccine
WO2022067010A1 (en) 2020-09-25 2022-03-31 Modernatx, Inc. Multi-proline-substituted coronavirus spike protein vaccines
WO2022099003A1 (en) 2020-11-06 2022-05-12 Sanofi Lipid nanoparticles for delivering mrna vaccines
CN116981692A (zh) * 2021-01-14 2023-10-31 翻译生物公司 递送mRNA编码的抗体的方法和组合物
US11524023B2 (en) 2021-02-19 2022-12-13 Modernatx, Inc. Lipid nanoparticle compositions and methods of formulating the same
AU2022237382A1 (en) 2021-03-15 2023-09-28 Modernatx, Inc. Therapeutic use of sars-cov-2 mrna domain vaccines
CN113289762A (zh) * 2021-05-07 2021-08-24 扬州三源机械有限公司 一种基于绿色环保的垃圾处理站用可回收垃圾分拣装置
WO2023025404A1 (en) 2021-08-24 2023-03-02 BioNTech SE In vitro transcription technologies
AU2022336209A1 (en) 2021-09-03 2024-01-18 CureVac SE Novel lipid nanoparticles for delivery of nucleic acids
CA3231003A1 (en) 2021-09-06 2023-03-09 Genmab A/S Antibodies capable of binding to cd27, variants thereof and uses thereof
TW202330610A (zh) 2021-10-08 2023-08-01 丹麥商珍美寶股份有限公司 結合至ｃｄ３０及ｃｄ３之抗體
WO2023069498A1 (en) 2021-10-22 2023-04-27 Senda Biosciences, Inc. Mrna vaccine composition
WO2023073228A1 (en) 2021-10-29 2023-05-04 CureVac SE Improved circular rna for expressing therapeutic proteins
WO2023092069A1 (en) 2021-11-18 2023-05-25 Modernatx, Inc. Sars-cov-2 mrna domain vaccines and methods of use
WO2023096858A1 (en) 2021-11-23 2023-06-01 Senda Biosciences, Inc. A bacteria-derived lipid composition and use thereof
WO2023144330A1 (en) 2022-01-28 2023-08-03 CureVac SE Nucleic acid encoded transcription factor inhibitors
WO2023205186A2 (en) * 2022-04-19 2023-10-26 Aegis Life, Inc. Dna therapeutic encoding an antibody or antigen binding fragment
WO2023227608A1 (en) 2022-05-25 2023-11-30 Glaxosmithkline Biologicals Sa Nucleic acid based vaccine encoding an escherichia coli fimh antigenic polypeptide
WO2024015890A1 (en) 2022-07-13 2024-01-18 Modernatx, Inc. Norovirus mrna vaccines

Family Cites Families (144)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4500707A (en) 1980-02-29 1985-02-19 University Patents, Inc. Nucleosides useful in the preparation of polynucleotides
US4458066A (en) 1980-02-29 1984-07-03 University Patents, Inc. Process for preparing polynucleotides
US5132418A (en) 1980-02-29 1992-07-21 University Patents, Inc. Process for preparing polynucleotides
US4415732A (en) 1981-03-27 1983-11-15 University Patents, Inc. Phosphoramidite compounds and processes
US4973679A (en) 1981-03-27 1990-11-27 University Patents, Inc. Process for oligonucleo tide synthesis using phosphormidite intermediates
US4668777A (en) 1981-03-27 1987-05-26 University Patents, Inc. Phosphoramidite nucleoside compounds
US4373071A (en) 1981-04-30 1983-02-08 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4401796A (en) 1981-04-30 1983-08-30 City Of Hope Research Institute Solid-phase synthesis of polynucleotides
US4897355A (en) 1985-01-07 1990-01-30 Syntex (U.S.A.) Inc. N[ω,(ω-1)-dialkyloxy]- and N-[ω,(ω-1)-dialkenyloxy]-alk-1-yl-N,N,N-tetrasubstituted ammonium lipids and uses therefor
US4737323A (en) 1986-02-13 1988-04-12 Liposome Technology, Inc. Liposome extrusion method
US5153319A (en) 1986-03-31 1992-10-06 University Patents, Inc. Process for preparing polynucleotides
US5047524A (en) 1988-12-21 1991-09-10 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
US5262530A (en) 1988-12-21 1993-11-16 Applied Biosystems, Inc. Automated system for polynucleotide synthesis and purification
FR2645866B1 (fr) 1989-04-17 1991-07-05 Centre Nat Rech Scient Nouvelles lipopolyamines, leur preparation et leur emploi
US5334761A (en) 1992-08-28 1994-08-02 Life Technologies, Inc. Cationic lipids
US5885613A (en) 1994-09-30 1999-03-23 The University Of British Columbia Bilayer stabilizing components and their use in forming programmable fusogenic liposomes
US5700642A (en) 1995-05-22 1997-12-23 Sri International Oligonucleotide sizing using immobilized cleavable primers
JP4335310B2 (ja) 1995-06-07 2009-09-30 ザ ユニバーシティ オブ ブリティッシュ コロンビア 疎水性脂質−核酸複合中間体を通して調製される脂質−核酸粒子、及び遺伝子移送のための使用
US5705385A (en) 1995-06-07 1998-01-06 Inex Pharmaceuticals Corporation Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US7422902B1 (en) 1995-06-07 2008-09-09 The University Of British Columbia Lipid-nucleic acid particles prepared via a hydrophobic lipid-nucleic acid complex intermediate and use for gene transfer
US5981501A (en) 1995-06-07 1999-11-09 Inex Pharmaceuticals Corp. Methods for encapsulating plasmids in lipid bilayers
US5744335A (en) 1995-09-19 1998-04-28 Mirus Corporation Process of transfecting a cell with a polynucleotide mixed with an amphipathic compound and a DNA-binding protein
IL140110A0 (en) * 2000-12-05 2002-02-10 Applied Research Systems Improvement of homogeneity and secretion of recombinant proteins in mammalian systems
CA2467363A1 (en) 2001-11-16 2003-06-12 Idec Pharmaceuticals Corporation Polycistronic expression of antibodies
DE10162480A1 (de) 2001-12-19 2003-08-07 Ingmar Hoerr Die Applikation von mRNA für den Einsatz als Therapeutikum gegen Tumorerkrankungen
JP4722481B2 (ja) 2002-06-28 2011-07-13 プロティバ バイオセラピューティクス リミテッド リポソーム製造方法および装置
JP4842821B2 (ja) 2003-09-15 2011-12-21 プロチバ バイオセラピューティクス インコーポレイティッド ポリエチレングリコール修飾脂質化合物およびその使用
EP1781593B1 (de) 2004-06-07 2011-12-14 Protiva Biotherapeutics Inc. Kationische lipide und verwendungsverfahren
JP4796062B2 (ja) 2004-06-07 2011-10-19 プロチバ バイオセラピューティクス インコーポレイティッド 脂質封入干渉ｒｎａ
JP2008506389A (ja) 2004-07-13 2008-03-06 セル ジェネシス インコーポレイテッド Ａａｖベクター組成物および免疫グロブリンの発現の増強のための方法ならびにその使用方法
DE102004042546A1 (de) 2004-09-02 2006-03-09 Curevac Gmbh Kombinationstherapie zur Immunstimulation
EP2395012B8 (de) 2005-11-02 2018-06-06 Arbutus Biopharma Corporation Modifizierte siRNA-Moleküle und Verwendungen davon
FR2904144A1 (fr) 2006-07-19 2008-01-25 St Microelectronics Rousset Procede de fabrication d'un wafer de semi-conducteur comprenant un filtre optique integre
US20080145413A1 (en) * 2006-12-19 2008-06-19 Steffen Panzner Lipids and lipid assemblies comprising transfection enhancer elements
DE102007001370A1 (de) * 2007-01-09 2008-07-10 Curevac Gmbh RNA-kodierte Antikörper
WO2009030254A1 (en) 2007-09-04 2009-03-12 Curevac Gmbh Complexes of rna and cationic peptides for transfection and for immunostimulation
WO2009046739A1 (en) 2007-10-09 2009-04-16 Curevac Gmbh Composition for treating prostate cancer (pca)
CA2910760C (en) 2007-12-04 2019-07-09 Muthiah Manoharan Targeting lipids
CA2710713C (en) 2007-12-27 2017-09-19 Protiva Biotherapeutics, Inc. Silencing of polo-like kinase expression using interfering rna
EP2224912B1 (de) 2008-01-02 2016-05-11 TEKMIRA Pharmaceuticals Corporation Verbesserte zusammensetzungen und verfahren zur freisetzung von nukleinsäuren
CA2711236A1 (en) 2008-01-02 2009-07-16 Alnylam Pharmaceuticals, Inc. Screening method for selected amino lipid-containing compositions
CA2721183C (en) 2008-04-11 2019-07-16 Alnylam Pharmaceuticals, Inc. Site-specific delivery of nucleic acids by combining targeting ligands with endosomolytic components
NZ588583A (en) 2008-04-15 2012-08-31 Protiva Biotherapeutics Inc Novel lipid formulations for nucleic acid delivery
WO2010037408A1 (en) 2008-09-30 2010-04-08 Curevac Gmbh Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof
WO2010042877A1 (en) 2008-10-09 2010-04-15 Tekmira Pharmaceuticals Corporation Improved amino lipids and methods for the delivery of nucleic acids
EP2365962B1 (de) 2008-11-07 2017-07-05 Massachusetts Institute of Technology Aminoalkohollipidoide und anwendungen davon
KR20220150995A (ko) 2008-11-10 2022-11-11 알닐람 파마슈티칼스 인코포레이티드 치료제 운반용 신규 지질 및 조성물
US9023820B2 (en) 2009-01-26 2015-05-05 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein C-III expression
SG10201402054UA (en) 2009-05-05 2014-09-26 Muthiah Manoharan Lipid compositions
CN104873464B (zh) 2009-06-10 2018-06-22 阿布特斯生物制药公司 改进的脂质制剂
WO2010147992A1 (en) 2009-06-15 2010-12-23 Alnylam Pharmaceuticals, Inc. Methods for increasing efficacy of lipid formulated sirna
WO2011000106A1 (en) 2009-07-01 2011-01-06 Protiva Biotherapeutics, Inc. Improved cationic lipids and methods for the delivery of therapeutic agents
CA2767129C (en) 2009-07-01 2015-01-06 Protiva Biotherapeutics, Inc. Compositions and methods for silencing apolipoprotein b
US8716464B2 (en) 2009-07-20 2014-05-06 Thomas W. Geisbert Compositions and methods for silencing Ebola virus gene expression
PT2506857T (pt) * 2009-12-01 2018-05-14 Translate Bio Inc Entrega de arnm para o acréscimo de proteínas e enzimas em doenças genéticas humanas
WO2011069529A1 (en) 2009-12-09 2011-06-16 Curevac Gmbh Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids
US20130017223A1 (en) 2009-12-18 2013-01-17 The University Of British Columbia Methods and compositions for delivery of nucleic acids
CA2799091A1 (en) 2010-05-12 2011-11-17 Protiva Biotherapeutics, Inc. Cationic lipids and methods of use thereof
WO2012000104A1 (en) 2010-06-30 2012-01-05 Protiva Biotherapeutics, Inc. Non-liposomal systems for nucleic acid delivery
HUE047796T2 (hu) 2010-07-06 2020-05-28 Glaxosmithkline Biologicals Sa RNS bevitele több immunútvonal bekapcsolására
US20130171241A1 (en) 2010-07-06 2013-07-04 Novartis Ag Liposomes with lipids having an advantageous pka-value for rna delivery
WO2012016184A2 (en) 2010-07-30 2012-02-02 Alnylam Pharmaceuticals, Inc. Methods and compositions for delivery of active agents
WO2012019168A2 (en) 2010-08-06 2012-02-09 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
US20120237975A1 (en) 2010-10-01 2012-09-20 Jason Schrum Engineered nucleic acids and methods of use thereof
WO2012075040A2 (en) 2010-11-30 2012-06-07 Shire Human Genetic Therapies, Inc. mRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES
WO2012116715A1 (en) 2011-03-02 2012-09-07 Curevac Gmbh Vaccination in newborns and infants
WO2012113413A1 (en) 2011-02-21 2012-08-30 Curevac Gmbh Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates
AU2012236099A1 (en) 2011-03-31 2013-10-03 Moderna Therapeutics, Inc. Delivery and formulation of engineered nucleic acids
EP2710136A4 (de) 2011-05-17 2015-01-21 Moderna Therapeutics Inc Manipulierte nukleinsäuren und verwendungsverfahren dafür für nicht-menschliche wirbeltiere
EP4074693A1 (de) 2011-06-08 2022-10-19 Translate Bio, Inc. Spaltbare lipide
SI2717893T1 (sl) 2011-06-08 2019-10-30 Translate Bio Inc Sestavki lipidnih nanodelcev in postopki za dostavo mRNA
US9464124B2 (en) 2011-09-12 2016-10-11 Moderna Therapeutics, Inc. Engineered nucleic acids and methods of use thereof
EP3384938A1 (de) 2011-09-12 2018-10-10 Moderna Therapeutics, Inc. Manipulierte nukleinsäuren und verfahren zur verwendung davon
WO2013039857A1 (en) 2011-09-12 2013-03-21 modeRNA Therapeutics Engineered nucleic acids and methods of use thereof
EP3682905B1 (de) 2011-10-03 2021-12-01 ModernaTX, Inc. Modifizierte nukleoside, nukleotide und nukleinsäuren und verwendungen davon
EA032088B1 (ru) 2011-10-27 2019-04-30 Массачусетс Инститьют Оф Текнолоджи Аминокислотные производные, функционализованные на n-конце, способные образовывать микросферы, инкапсулирующие лекарственное средство
EP2791159A4 (de) 2011-12-14 2015-10-14 Moderna Therapeutics Inc Modifizierte nucleinsäuren und akutbehandlungsverwendungen davon
EP2791364A4 (de) 2011-12-14 2015-11-11 Moderna Therapeutics Inc Verfahren zur reaktion auf eine biologische bedrohung
RS63244B1 (sr) 2011-12-16 2022-06-30 Modernatx Inc Kompozicije modifikovane mrna
CA2859691A1 (en) 2011-12-21 2013-06-27 Moderna Therapeutics, Inc. Methods of increasing the viability or longevity of an organ or organ explant
WO2013101690A1 (en) 2011-12-29 2013-07-04 modeRNA Therapeutics Modified mrnas encoding cell-penetrating polypeptides
WO2013120499A1 (en) 2012-02-15 2013-08-22 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly (a) sequence or a polyadenylation signal for increasing the expression of an encoded pathogenic antigen
WO2013120497A1 (en) 2012-02-15 2013-08-22 Curevac Gmbh Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded therapeutic protein
KR102239887B1 (ko) 2012-02-24 2021-04-13 아뷰터스 바이오파마 코포레이션 트리알킬 양이온성 지질 및 그의 사용 방법
US9572897B2 (en) 2012-04-02 2017-02-21 Modernatx, Inc. Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins
CN108949772A (zh) 2012-04-02 2018-12-07 现代泰克斯公司 用于产生与人类疾病相关的生物制剂和蛋白质的修饰多核苷酸
US20150050354A1 (en) 2012-04-02 2015-02-19 Moderna Therapeutics, Inc. Modified polynucleotides for the treatment of otic diseases and conditions
US9283287B2 (en) 2012-04-02 2016-03-15 Moderna Therapeutics, Inc. Modified polynucleotides for the production of nuclear proteins
US10501512B2 (en) 2012-04-02 2019-12-10 Modernatx, Inc. Modified polynucleotides
US20140275229A1 (en) 2012-04-02 2014-09-18 Moderna Therapeutics, Inc. Modified polynucleotides encoding udp glucuronosyltransferase 1 family, polypeptide a1
EP2833892A4 (de) 2012-04-02 2016-07-20 Moderna Therapeutics Inc Modifizierte polynukleotide zur herstellung von in der onkologie nützlichen proteinen und peptiden
JP6561378B2 (ja) 2012-06-08 2019-08-21 トランスレイト バイオ， インコーポレイテッド 非肺標的細胞へのｍＲＮＡの経肺送達
ES2921623T3 (es) 2012-11-26 2022-08-30 Modernatx Inc ARN modificado terminalmente
US9636301B2 (en) 2012-12-04 2017-05-02 Arbutus Biopharma Corporation In vitro release assay for liposome encapsulated vincristine
EP2929035A1 (de) 2012-12-07 2015-10-14 Shire Human Genetic Therapies, Inc. Lipidnanopartikel zur freisetzung von mrna
US20150315541A1 (en) 2012-12-13 2015-11-05 Moderna Therapeutics, Inc. Modified polynucleotides for altering cell phenotype
EP2931319B1 (de) 2012-12-13 2019-08-21 ModernaTX, Inc. Modifizierte nukleinsäuremoleküle und deren verwendungen
WO2014113089A2 (en) 2013-01-17 2014-07-24 Moderna Therapeutics, Inc. Signal-sensor polynucleotides for the alteration of cellular phenotypes
EP2968397A4 (de) 2013-03-12 2016-12-28 Moderna Therapeutics Inc Diagnose und behandlung von fibrose
WO2014159813A1 (en) 2013-03-13 2014-10-02 Moderna Therapeutics, Inc. Long-lived polynucleotide molecules
US10258698B2 (en) 2013-03-14 2019-04-16 Modernatx, Inc. Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions
AU2014239184B2 (en) 2013-03-14 2018-11-08 Translate Bio, Inc. Methods and compositions for delivering mRNA coded antibodies
WO2014152030A1 (en) 2013-03-15 2014-09-25 Moderna Therapeutics, Inc. Removal of dna fragments in mrna production process
EP3578663A1 (de) 2013-03-15 2019-12-11 ModernaTX, Inc. Herstellungsverfahren zur herstellung von rna-transkripten
US8980864B2 (en) 2013-03-15 2015-03-17 Moderna Therapeutics, Inc. Compositions and methods of altering cholesterol levels
WO2014144039A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Characterization of mrna molecules
US20160017313A1 (en) 2013-03-15 2016-01-21 Moderna Therapeutics, Inc. Analysis of mrna heterogeneity and stability
WO2014144767A1 (en) 2013-03-15 2014-09-18 Moderna Therapeutics, Inc. Ion exchange purification of mrna
US11377470B2 (en) 2013-03-15 2022-07-05 Modernatx, Inc. Ribonucleic acid purification
HRP20211563T1 (hr) 2013-07-11 2022-01-07 Modernatx, Inc. Pripravci koji sadrže sintetske polinukleotide koji kodiraju proteine srodne crispr-u i sintetske sgrna, te postupci njihove uporabe
WO2015011633A1 (en) 2013-07-23 2015-01-29 Protiva Biotherapeutics, Inc. Compositions and methods for delivering messenger rna
CA2923029A1 (en) 2013-09-03 2015-03-12 Moderna Therapeutics, Inc. Chimeric polynucleotides
EP3041938A1 (de) 2013-09-03 2016-07-13 Moderna Therapeutics, Inc. Kreisförmige polynukleotide
WO2015048744A2 (en) 2013-09-30 2015-04-02 Moderna Therapeutics, Inc. Polynucleotides encoding immune modulating polypeptides
EP3052479A4 (de) 2013-10-02 2017-10-25 Moderna Therapeutics, Inc. Polynukleotidmoleküle und verwendungen davon
WO2015051169A2 (en) 2013-10-02 2015-04-09 Moderna Therapeutics, Inc. Polynucleotide molecules and uses thereof
AU2014337156A1 (en) 2013-10-18 2016-05-12 Modernatx, Inc. Compositions and methods for tolerizing cellular systems
EA201690588A1 (ru) 2013-10-22 2016-09-30 Шир Хьюман Дженетик Терапис, Инк. Доставка мрнк в цнс и ее применение
US20170173128A1 (en) 2013-12-06 2017-06-22 Moderna TX, Inc. Targeted adaptive vaccines
US20150167017A1 (en) 2013-12-13 2015-06-18 Moderna Therapeutics, Inc. Alternative nucleic acid molecules and uses thereof
CA2927862C (en) 2013-12-30 2024-01-23 Curevac Ag Artificial nucleic acid molecules
US20170002060A1 (en) 2014-01-08 2017-01-05 Moderna Therapeutics, Inc. Polynucleotides for the in vivo production of antibodies
ES2754239T3 (es) 2014-03-12 2020-04-16 Curevac Ag Combinación de vacunación y agonistas de OX40
SG11201608798YA (en) 2014-04-23 2016-11-29 Modernatx Inc Nucleic acid vaccines
ES2727776T3 (es) 2014-06-10 2019-10-18 Curevac Ag Método para mejorar la producción de ARN
US20170136132A1 (en) 2014-06-19 2017-05-18 Moderna Therapeutics, Inc. Alternative nucleic acid molecules and uses thereof
WO2015196128A2 (en) 2014-06-19 2015-12-23 Moderna Therapeutics, Inc. Alternative nucleic acid molecules and uses thereof
EP3201338B1 (de) 2014-10-02 2021-11-03 Arbutus Biopharma Corporation Zusammensetzungen und verfahren zur ausschaltung einer hepatitis-b-virus-genexpression
WO2016071857A1 (en) 2014-11-07 2016-05-12 Protiva Biotherapeutics, Inc. Compositions and methods for silencing ebola virus expression
EP4324473A2 (de) 2014-11-10 2024-02-21 ModernaTX, Inc. Optimierung multiparametrischer nukleinsäuren
EP3041948B1 (de) 2014-11-10 2019-01-09 Modernatx, Inc. Alternative nukleinsäuremoleküle mit reduziertem uracilgehalt und verwendungen davon
US20180000953A1 (en) 2015-01-21 2018-01-04 Moderna Therapeutics, Inc. Lipid nanoparticle compositions
WO2016118725A1 (en) 2015-01-23 2016-07-28 Moderna Therapeutics, Inc. Lipid nanoparticle compositions
US20180245077A1 (en) 2015-03-20 2018-08-30 Protiva Biotherapeutics, Inc. Compositions and methods for treating hypertriglyceridemia
WO2016164762A1 (en) 2015-04-08 2016-10-13 Moderna Therapeutics, Inc. Polynucleotides encoding low density lipoprotein receptor egf-a and intracellular domain mutants and methods of using the same
WO2016183366A2 (en) 2015-05-12 2016-11-17 Protiva Biotherapeutics, Inc. Compositions and methods for silencing expression of hepatitis d virus rna
WO2016197133A1 (en) 2015-06-04 2016-12-08 Protiva Biotherapeutics, Inc. Delivering crispr therapeutics with lipid nanoparticles
WO2016197132A1 (en) 2015-06-04 2016-12-08 Protiva Biotherapeutics Inc. Treating hepatitis b virus infection using crispr
EP3307305A4 (de) 2015-06-10 2019-05-22 Modernatx, Inc. Gerichtete adaptive impfstoffe
WO2017019891A2 (en) 2015-07-29 2017-02-02 Protiva Biotherapeutics, Inc. Compositions and methods for silencing hepatitis b virus gene expression
EP3350333B1 (de) 2015-09-17 2021-10-27 ModernaTX, Inc. Polynukleotide mit stabilisierendem schwanzbereich
WO2017049286A1 (en) 2015-09-17 2017-03-23 Moderna Therapeutics, Inc. Polynucleotides containing a morpholino linker
US20190054112A1 (en) 2015-09-18 2019-02-21 Moderna Therapeutics, Inc. Polynucleotide formulations for use in the treatment of renal diseases
USD787703S1 (en) 2016-04-26 2017-05-23 Curevac Ag Inlay for a culture plate

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2014152774A1 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11421038B2 (en) 2007-01-09 2022-08-23 Curevac Ag RNA-coded antibody
WO2014152774A1 (en) 2013-03-14 2014-09-25 Shire Human Genetic Therapies, Inc. Methods and compositions for delivering mrna coded antibodies

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